Appendix 7 – Methods for Benthic Monitoring

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Appendix 7 – Methods for Benthic Monitoring

by Contact on 30 October 2026

Appendix 7 – Methods for Benthic Monitoring

This Appendix details the methods for benthic monitoring required in Criterion 2.5. It also details the method for determining the distance between the site and seagrass habitats (required in Criterion 2.2), and copper monitoring (required in Criterion 2.11) for sites using copper nets or copper-treated nets.

Section

7.1 Introduction

7.2 Method for cage culture in marine/brackish waters

7.3 Method for cage culture in freshwater lakes/reservoirs

7.4 Method for suspended marine mollusc systems

7.5 Method for freshwater systems discharging into rivers

7.6 Method for determining the distance between the site and seagrass habitats

7.7 Method for cage culture in marine/brackish waters using copper nets or copper-treated nets

7.8 Methods for field analysis of abiotic Indicators

7.1 Introduction

For cage culture and suspended marine mollusc culture systems (Indicators 2.5.1 and 2.5.2), the ASC method for benthic monitoring requires sampling at predefined zones to determine benthic status. The method is based on a three-level approach in which a site conducts increasingly more detailed benthic monitoring if the results of Level 1 or Level 2 monitoring do not demonstrate an ‘Acceptable’ benthic status. Conversely, a site that demonstrates an ‘Acceptable’ benthic status in Level 1 or Level 2 does not need to conduct a higher level of monitoring. Sites may decide to start monitoring at any of the three levels. The method uses five benthic Ecological Quality Status (EQS) categories that are numerically defined using a range of abiotic and biotic Indicator thresholds (see Table 1 and Table 2). An EQS Category is assigned to the monitoring results of each sampling zone which determines the benthic status of a site.

In the case of cage culture in marine/brackish waters using copper nets or copper-treated nets (Indicator 2.11.11), the method requires additional benthic monitoring to determine the copper level in sediments.

Sites producing Seabass, seabream and meagre are required to determine the distance to seagrass habitat from the zone 1 boundary (Indicator 2.2.5) outlined in Figure 1.

For freshwater systems discharging into rivers (Indicator 2.5.4), the ASC method for benthic monitoring requires sampling upstream and downstream from the site outflow point to determine the presence, composition, abundance and diversity of invertebrate fauna, and to categorise the benthic status upstream and downstream.

ASC has benchmarked the ASC Farm Standard methods for benthic monitoring against applicable regulatory requirements. Outcomes of the benchmark are available via the ASC Programme Centre.

Table 1. Abiotic Indicator thresholds for the EQS categories used by the methods for cage culture and suspended marine mollusc systems.

Abiotic Indicators

EQS Category Thresholds

High

Good

Moderate

Poor

Bad

Total Free Sulphide (S2-UV; µM)

75

250

500

1100

> 1100

Total Free Sulphide (S2-ISE; µM)

750

1500

3000

6000

> 6000

Redox potential (Eh)

100

0

-100

-150

<-150

Total Ammonia Nitrogen (TAN; mg/L)

< 1.9 at pH 7, 20°C

1.9 at pH 7, 20°C129129At pH 7 and 20oC. For other pH and/or temperature see applicable thresholds in Section 7.8.5.1.

>1.9 at pH 7, 20°C

Table 2. Biotic Indicator thresholds for the EQS categories used by the methods for cage culture and suspended marine mollusc systems.

Biotic Indicators

EQS Category Thresholds

High

Good

Moderate

Poor

Bad

Richness (% of max S)

>80

50

35

15

<15

Opportunistic Taxa (GrV; %)

<20

40

60

80

>80

Polychaete/Amphipod Ratio (BPOFA)

<0.031

0.126

0.187

0.237

>0.237

AZTI’s Marine Biotic Index (AMBI)

<1.2

3.0

3.9

4.8

>4.8

Multivariate AMBI (M-AMBI)

>0.83

0.59

0.47

0.35

<0.35

Benthic Habitat Quality (BHQ)

>8

6

4

2

<2

Simplified Richness (S50)

>16

11.7

7.5

5.4

<5.4

Benthic Quality Index (BQI)

>16.0

12.0

8.0

4.0

<4.0

Benthic Quality Index (BQI-family)

>20.8

9.2

5.7

1.9

<1.9

BENTIX

>0.67

0.5

0.42

0.33

<0.33

Norwegian Quality Index (NQI1)

>0.86

0.68

0.43

0.20

<0.20

Norwegian Sensitivity Index (NSI2012)

> 27.4

23.1

18.8

10.4

< 10.4

Indicator Species Index (ISI2012)

>9.6

7.5

6.2

4.5

<4.5

Enrichment Stage (ES)

1

2

3 to 4

4 to 5

6 to 7

7.2 Method for Cage Culture in Marine/Brackish Waters (Indicators 2.5.1 and 2.5.2) 

7.2.1 Sampling Method

Level 1: This monitoring level provides a rapid assessment of benthic impacts in the direction of greatest risk (i.e. the direction of the predominant current) and employs practical abiotic Indicators.

  1. Sediment sampling shall be conducted in duplicate within each of the sampling zones outlined in Figure 1: 0-30, 31-100, 101-150, and at 500 metres from the edge of the cage array, in one transect situated in the direction of the predominant current. The 500-metre sampling location represents the reference zone.

  2. The samples shall be analysed immediately on the survey vessel for:

    • Total free sulphide (S2-): in triplicate for each sample (six analyses in total for each sampling location), either using the ion-selective electrode method (S2-ISE) or the UV spectrophotometry method (S2-UV)130130From May 2028, UV spectrophotometry will be the only acceptable method for total free sulphide analysis. outlined in Section 7.8; and

    • Redox potential (Eh): single measure for each sample (two analyses in total for each sampling location), using the method outlined in Section 7.8.

  3. With the results of the analysis of the samples, the EQS Category shall be calculated to determine the benthic status of each sampling zone following the steps outlined in Section 7.2.2.

If the EQS Category results in:

  • An ‘Acceptable’ benthic status for each sampling zone; no additional sampling is required.

  • An ‘Unacceptable’ benthic status for any sampling zone; either Level 2 or Level 3 monitoring shall be conducted immediately.

Level 2: This monitoring level provides a two-dimensional spatial analysis of benthic conditions using abiotic Indicators.

  1. Sediment sampling shall be conducted as for Level 1 but in three additional transect directions according to Figure 1.

  2. The samples shall be analysed immediately on the survey vessel for:

    • Total free sulphide (S2-): in triplicate for each sample (6 analyses in total for each sampling location, and 24 analyses in total for each sampling zone), either using the ion selective electrode method (S2-ISE) or the UV spectrophotometry method (S2-UV) 130130From May 2028, UV spectrophotometry will be the only acceptable method for total free sulphide analysis. outlined in Section 7.8; and

    • Redox potential (Eh): single measure for each sample (2 analyses in total for each sampling location and 8 in total for each sampling zone) using the method outlined in Section 7.8.

  3. With the results of the analysis of the samples, the EQS Category shall be calculated to determine the benthic status of each sampling zone following the steps outlined in Section 7.2.2.

If the EQS Category results in:

  • An ‘Acceptable’ benthic status for each sampling zone; no additional sampling is required.

  • An ‘Unacceptable’ benthic status for any sampling zone; Level 3 monitoring shall be conducted immediately.

Level 3: This monitoring level provides direct information on the impact of the site on the benthic macrofauna community.

  1. Sediment samples shall be collected in duplicate at the sampling locations outlined for Level 2, at a minimum, on the two transects in the direction of the predominant current, according to Figure 1.

  2. The samples shall be screened through a 1.0mm mesh, and all organisms shall be preserved for taxonomic analysis.

  3. The samples shall be analysed for three biotic Indicators from Table 2.

  4. With the results of the analysis of the samples, the EQS Category shall be calculated to determine the benthic status of each sampling zone following the steps outlined in Section 7.2.2.

If the EQS Category results in:

  • An ‘Acceptable’ benthic status for each sampling zone; no additional sampling is required.

  • An ‘Unacceptable’ benthic status for any sampling zones; the site does not conform with Indicator 2.5.2.

Zone 1

Zone 2

Zone 3

30 m

100 m

150 m

500 m Reference Zone

Predominant Current

Figure 1. Cage Systems in Marine/Brackish – Sampling zones (shaded areas) and sampling locations (dots) required for Level 1 (●), 2 (● and ○) and 3 (● and ○) monitoring. Sampling locations are established on transects within the boundaries of sampling zones 1, 2 and 3 and at the 500 metres reference zone. Sample stations in zones 1, 2 and 3 can be located at any distance within the zone (from the cage array). For example, the zone 2 sample station can be located anywhere within 30 m and 100 m from the cage array. Figure 1 presents an example of the sampling locations inside the boundaries of zones 1, 2 and 3.

Sampling Timing

Sampling shall be conducted during the period when the benthic impact is expected to be highest. Sites shall provide information on when the maximum impact on the benthos is predicted to occur. Based on this information:

  • Sampling shall be conducted during the final year of each production cycle within 30 days:

    • after peak feeding; or,

    • after peak biomass; or

    • after maximum annual bottom water temperature.

  • In the case of multiple peaks in feeding/biomass occurring in any year, sampling shall be conducted annually within 30 days after the maximum annual bottom water temperature.

After three years of demonstrating conformance with Indicator 2.5.2, sites with multiple peaks in feeding/biomass occurring in any year may reduce sampling to once per certification cycle (i.e. every three years) as long as there have been no significant changes to farming practices and thus that previously determined benthic status provides a credible proxy for future performance.

7.2.2 Conformance Framework

A numerical scoring method is used to combine all Indicator data collected within each sampling zone, which allows for determining the EQS Category and benthic status of the site as required by Indicator 2.5.2.

The steps to calculate the EQS Category for each sampling zone are as follows:

  1. The mean value for each Indicator shall be calculated for each sampling zone and compared with Table 1, in the case of abiotic Indicators, or Table 2, in the case of biotic Indicators, to determine the EQS Category that applies to each Indicator (i.e. ‘High’, ‘Good’, ‘Moderate’, ‘Poor’ and ‘Bad’).

  2. The determined EQS Category for each Indicator shall be compared with Table 3 to assign an Indicator EQS Score to each sampling zone.

    Table 3. Indicator EQS Scores associated with the EQS Categories.

    EQS Category

    High

    Good

    Moderate

    Poor

    Bad

    Indicator EQS Score

    1

    2

    3

    4

    5

  3. The overall Zone EQS Score shall then be calculated by combining the Indicator EQS Scores using the following formulas:

    • Level 1 and 2 monitoring:

      • S2-UV and Eh Indicators:

         EQS Scorezone=EQSS2−uv×0.75+EQSEh×0.25 {EQS Score }_{zone}={EQS}_{{{S}^{2-}}_{uv}}times 0.75+{EQS}_{Eh}times 0.25 

      • S2-ISE and Eh Indicators:

         EQS Scorezone=EQSS2−ISE+EQSEh2 {EQS Score}_{zone} = frac{{EQS}_{{S^{2 -}}_{ISE}} + {EQS}_{Eh}}{2} 

    • Level 3 monitoring:

      • Three biotic Indicators: A, B and C.

         EQS Scorezone=EQSA+EQSB+EQSC3 {EQS Score}_{zone} = frac{{EQS}_{A} + {EQS}_{B} + {EQS}_{C}}{3} 

  4. The resulting Zone EQS Score determines the EQS Category to be applied to each sampling zone, as shown in Table 4:

    Table 4. EQS Categories associated with the Zone EQS Scores

    Zone EQS Score

    1.0

    1.1 to 2.0

    2.1 to 3.0

    3.1 to 4.0

    >4.0

    EQS Category

    High

    Good

    Moderate

    Poor

    Bad

  5. The EQS Category obtained from Table 4 shall be compared with Table 5 to determine the benthic status of the sampling zone.  The EQS Category of the reference zone determines the EQS Category for ‘Acceptable’ benthic status for each sampling zone.

Table 5. EQS Categories for ‘Acceptable’ benthic status.

EQS Category Reference Zone

Sampling Zone

EQS Category for ‘Acceptable’ Benthic Status

‘High’ EQS Category

Zone 1

At least ‘Moderate’ EQS Category shall be achieved by 30m

Zone 2

At least ‘Good’ EQS Category shall be achieved by 100m

Zone 3

‘High’ EQS Category shall be achieved by 150m

‘Good’ EQS Category

Zone 1

At least ‘Moderate’ EQS Category shall be achieved by 30m

Zones 2 and 3

‘Good’ EQS Category shall be achieved at 100 and 150m

‘Moderate’ EQS Category

Zone 1, 2 and 3

At least ‘Moderate’ EQS Category shall be achieved in all zones

‘Poor’ or ‘Bad’ EQS Category

Not an acceptable EQS Category

7.3 Method for Cage Culture in Freshwater Lakes/Reservoirs (Indicators 2.5.1 and 2.5.2)

7.3.1 Sampling Method

Level 1: This monitoring level provides a rapid assessment of benthic impacts in the direction of greatest risk (i.e. the direction of the predominant current) and employs practical abiotic Indicators.

  1. Sediment sampling shall be conducted in duplicate within each of the sampling zones outlined in Figure 2: 0-30, 31-100 and at 150 metres from the edge of the cage array, in one transect situated in the direction of the predominant current. The 150-metre sampling location represents the reference zone.

  2. The samples shall be analysed immediately on the survey vessel for:

    • Redox potential (Eh): single measure for each sample (two analyses in total for each sampling location), using the method outlined in Section 7.8; and

    • Total Ammonia Nitrogen (TAN): single measure for each sample (two analyses in total for each sampling location), using the method outlined in Section 7.8. TAN analysis requires information on the sample temperature and pH. The method to analyse pH is also outlined in Section 7.8.

  3. With the results of the analysis of the samples, the EQS Category shall be calculated to determine the benthic status of each sampling zone following the steps outlined in Section 7.3.2.

If the EQS Category results in:

  • An ‘Acceptable’ benthic status for each sampling zone; no additional sampling is required.

  • An ‘Unacceptable’ benthic status for any sampling zone; either Level 2 or Level 3 monitoring shall be conducted immediately.

Level 2: This monitoring level provides a two-dimensional spatial analysis of benthic conditions using abiotic Indicators.

  1. Sediment sampling shall be conducted as for Level 1 but in three additional transect directions according to Figure 2.

  2. The samples shall be analysed immediately on the survey vessel for:

    • Redox potential (Eh): single measure for each sample (two analyses in total for each sampling location), using the method outlined in Section 7.8; and

    • Total Ammonia Nitrogen (TAN): single measure for each sample (two analyses in total for each sampling location), using the method outlined in Section 7.8. TAN analysis requires information on the sample temperature and pH. The method to analyse pH is also outlined in Section 7.8.

  3. With the results of the analysis of the samples, the EQS Category shall be calculated to determine the benthic status of each sampling zone following the steps outlined in Section 7.3.2.

If the EQS Category results in:

  • An ‘Acceptable’ benthic status for each sampling zone; no additional sampling is required.

  • An ‘Unacceptable’ benthic status for any sampling zone; Level 3 monitoring shall be conducted immediately.

Level 3: This monitoring level provides direct information on the impact of the site on the benthic macrofauna community.

  1. Sediment samples shall be collected in duplicate at the sampling locations outlined for Level 2, at a minimum, on the two transects in the direction of the predominant current, according to Figure 2.

  2. The samples shall be screened through a 1.0mm mesh, and all organisms preserved for taxonomic analysis.

  3. The samples shall be analysed for three biotic Indicators from Table 2.

  4. With the results of the analysis of the samples, the EQS Category shall be calculated to determine the benthic status of each sampling zone following the steps outlined in Section 7.3.2.

If the EQS Category results in:

  • An ‘Acceptable’ benthic status for each sampling zone; no additional sampling is required.

  • An ‘Unacceptable’ benthic status for any sampling zones; the site does not conform with Indicator 2.5.2131131Demonstration that the status is ‘Acceptable’ is not required until the mandatory date of the ASC Farm Standard in May 2028..

Zone 1

Zone 2

30 m

100 m

150 m Reference Zone

Predominant Current

Figure 2. Cage Systems in Freshwater Lakes/Reservoirs – Sampling zones (shaded areas) and sampling locations (dots) required for Level 1 (●), 2 (● and ○) and 3 (● and ○) monitoring. Sampling locations are established on transects within the boundaries of sampling zones 1 and 2 and at the 150 metre reference zone. Sample stations in zones 1 and 2 can be located at any distance within the zone (from the cage array). The reference station shall be located 150 m from the cage array. For example, the zone 2 sample station can be located anywhere within 30 m and 100 m from the cage array. Figure 2 presents an example of the sampling locations inside the boundaries of zones 1 and 2.

Sampling Timing

Sampling shall be conducted during the period when the benthic impact is expected to be highest. Sites shall provide information on when the maximum impact on the benthos is predicted to occur. Based on this information:

  • Sampling shall be conducted during the final year of each production cycle within 30 days:

  • after peak feeding; or,

  • after peak biomass; or

  • after maximum annual bottom water temperature.

  • In the case of multiple peaks in feeding/biomass occurring in any year, sampling shall be conducted annually within 30 days after the maximum annual bottom water temperature.

After three years of demonstrating conformance with Indicator 2.5.2, sites with multiple peaks in feeding/biomass occurring in any year may reduce sampling to once per certification cycle (i.e. every three years) as long as there have been no significant changes to farming practices and thus that previously determined benthic status provides a credible proxy for future performance.

7.3.2 Conformance Framework

A numerical scoring method is used to combine all Indicator data collected within each sampling zone, which allows for determining the EQS Category and benthic status of the site as required by Indicator 2.5.2.

The steps to calculate the EQS Category for each sampling zone are as follows:

  1. The mean value for each Indicator shall be calculated for each sampling zone and compared with Table 1, in the case of abiotic Indicators, or Table 2, in the case of biotic Indicators, to determine the EQS Category that applies to each Indicator (i.e. ‘High’, ‘Good’, ‘Moderate’, ‘Poor’ and ‘Bad’).

  2. The determined EQS Category for each Indicator shall be compared with Table 6 to assign an Indicator EQS Score within each sampling zone.

    Table 6. Indicator EQS Scores associated with the EQS Categories.

    EQS Category

    High

    Good

    Moderate

    Poor

    Bad

    Eh Indicator EQS Score

    1

    2

    3

    4

    5

    TAN Indicator EQS Score

    1

    3

    5

  3. The overall Zone EQS Score shall then be calculated by combining the Indicator EQS Scores using the following equations:

    • Level 1 and 2 monitoring:

      • TAN and Eh Indicators:

         EQS Scorezone=EQSTAN+EQSEh2 {EQS Score}_{zone} = frac{{EQS}_{TAN} + {EQS}_{Eh}}{2} 

    • Level 3 monitoring:

      • Three biotic Indicators: A, B and C.

         EQS Scorezone=EQSA+EQSB+EQSC3 {EQS Score}_{zone} = frac{{EQS}_{A} + {EQS}_{B} + {EQS}_{C}}{3} 

  4. The resulting Zone EQS Score determines the EQS Category to be applied to each sampling zone, as shown in Table 7:

    Table 7. EQS Categories associated with the Zone EQS Scores

    Zone EQS Score

    1.0

    1.1 to 2.0

    2.1 to 3.0

    3.1 to 4.0

    >4.0

    EQS Category

    High

    Good

    Moderate

    Poor

    Bad

  5. The EQS Category obtained from Table 7 shall be compared with Table 8 to determine the benthic status of the sampling zone.  The EQS Category of the reference zone determines the EQS Category for ‘Acceptable’ benthic status for each sampling zone.

Table 8 EQS Categories for ‘Acceptable’ benthic status

EQS Category Reference Zone

Sampling Zone

EQS Category for ‘Acceptable’ Benthic Status

‘High’ or ‘Good’ EQS Category

Zone 1

At least ‘Moderate’ EQS Category shall be achieved by 30m

‘Moderate’ EQS Category

Zone 1 and 2

At least ‘Moderate’ EQS Category shall be achieved in both zones

‘Poor”‘or ‘Bad’ EQS Category

Not an acceptable EQS Category

7.4 Method for Suspended Marine Molluscs Systems (Indicators 2.5.1 and 2.5.2)

7.4.1 Sampling Method

Level 1: This monitoring level provides a rapid assessment of benthic impacts in the direction of greatest risk (i.e. the direction of the predominant current) and uses practical abiotic Indicators.

  1. Sediment sampling shall be conducted in duplicate in each of the five sampling locations situated 10m apart along one transect located in the direction of the predominant current, with the third sampling location (i.e. the middle of the five locations) being situated inside the edge of the site boundary, as outlined in Figure 3132132ASC will develop sampling guidance for circumstances in which multiple marine mollusc sites have shared boundaries..

  2. The samples shall be analysed immediately on the survey vessel for:

    • Total free sulphide (S2-): in triplicate for each bottom sample (six analyses in total for each sampling location), either using the ion-selective electrode method (S2-ISE) or the UV spectrophotometry method (S2-UV)133133From May 2028, UV spectrophotometry will be the only acceptable method for total free sulphide analysis. outlined in Section 7.8; and

    • Redox potential (Eh): single measure at each sampling location (two analyses in total for each sampling location), using the method outlined in Section 7.8.

  3. With the results of the analysis of the samples, the EQS Category shall be calculated to determine the benthic status of each sampling zone following the steps outlined in Section 7.4.2.

If the EQS Category results in:

  • An ‘Acceptable’ benthic status for each sampling zone; no additional sampling is required.

  • An ‘Unacceptable’ benthic status for any sampling zone; either Level 2 or Level 3 monitoring shall be conducted immediately.

Level 2: This monitoring level provides a two-dimensional spatial analysis of benthic conditions using abiotic Indicators.

  1. Sediment sampling shall be conducted as for Level 1 but in three additional transect directions according to Figure 3.

  2. The samples shall be analysed immediately on the survey vessel for:

    • Total free sulphide (S2-): in triplicate for each sample (six analyses at each station and 24 analyses in total for each sampling distance), either using the ion selective electrode method (S2-ISE) or the UV spectrophotometry method (S2-UV)133133From May 2028, UV spectrophotometry will be the only acceptable method for total free sulphide analysis. outlined in Section 7.8.

    • Redox potential (Eh): single measure for each sediment sample (two analyses at each station and eight in total for each sampling distance) using the method outlined in Section 7.8.

  3. With the results of the analysis of the samples, the EQS Category shall be calculated to determine the benthic status of each sampling zone following the steps outlined in Section 7.4.2.

If the EQS Category results in:

  • An ‘Acceptable’ benthic status for each sampling zone; no additional sampling is required.

  • An ‘Unacceptable’ benthic status for any sampling zone; Level 3 monitoring shall be conducted immediately.

Level 3: This monitoring level provides direct information on the impact of the site on the benthic macrofauna community.

  1. Sediment samples shall be collected in duplicate at the sampling locations outlined for Level 2, at a minimum, on the two transects in the direction of the predominant current, according to Figure 3.

  2. The samples shall be screened through a 1.0mm mesh, and all organisms preserved for taxonomic analysis.

  3. The samples shall be analysed for three biotic Indicators from Table 2.

  4. With the results of the analysis of the samples, the EQS Category shall be calculated to determine the benthic status of each sampling zone following the steps outlined in Section 7.4.2.

If the EQS Category results in:

  • An ‘Acceptable’ benthic status for each sampling zone; no additional sampling is required.

  • An ‘Unacceptable’ benthic status for any sampling zones; the site does not conform with Indicator 2.5.2.

Predominant Current

Site Boundary

Inside Site Boundary

Outside Site Boundary

Figure 3. Suspended Marine Molluscs Systems – Sampling locations (dots) per zone (inside and outside site boundary) required for Level 1 (●), 2 (● and ○) and 3 (● and ○). Sampling locations on each transect are 10m apart, with the third sampling location (i.e. the middle of the five locations) being situated inside the edge of the site boundary.

Sampling Timing

  • Sampling shall be conducted during the final year of each production cycle within 30 days after peak biomass.

  • In the case of multiple peaks in biomass occurring in any year, sampling shall be conducted annually within 30 days after the maximum annual bottom water temperature.

Sites may reduce the sampling frequency to once per certification cycle (i.e. every three years) if the EQS Category for Benthic Status inside the site boundary is ‘Good’ or High’ and the EQS Category for Benthic Status outside the site boundary is at least ‘Moderate’.

The reduction in sampling frequency shall be permitted as long as there have been no significant changes to farming practices and therefore the previously determined benthic status provides a credible proxy for future performance.

7.4.2 Conformance Framework

A numerical scoring method is used to combine all Indicator data collected within each sampling zone, which allows for determining the EQS Category and benthic status of the sampling zones surrounding the site as required by Indicator 2.5.2.

The steps to calculate the EQS Category for each sampling zone are as follows:

  1. The mean value for each Indicator shall be calculated for each sampling zone and compared with Table 1, in the case of abiotic Indicators, or Table 2, in the case of biotic Indicators, to determine the EQS Category that applies to each Indicator (i.e. ‘High’, ‘Good’, ‘Moderate’, ‘Poor’ and ‘Bad’).

  2. The determined EQS Category for each Indicator shall be compared with Table 9 to assign an Indicator EQS Score within each sampling zone.

    Table 9. Indicator EQS Scores associated with the EQS Categories.

    EQS Category

    High

    Good

    Moderate

    Poor

    Bad

    Indicator EQS Score

    1

    2

    3

    4

    5

  3. The overall Zone EQS Score shall then be calculated by combining the Indicator EQS scores using the following equations:

    • Level 1 and 2 monitoring:

      • S2-UV and Eh Indicators:

         EQS Scorezone=EQSS2−uv×0.75+EQSEh×0.25 {EQS Score }_{zone}={EQS}_{{{S}^{2-}}_{uv}}times 0.75+{EQS}_{Eh}times 0.25 

      • S2-ISE and Eh Indicators:

         EQS Scorezone=EQSS2−ISE+EQSEh2 {EQS Score}_{zone} = frac{{EQS}_{{S^{2 -}}_{ISE}} + {EQS}_{Eh}}{2} 

    • Level 3 monitoring:

      • Three biotic Indicators: A, B and C.

         EQS Scorezone=EQSA+EQSB+EQSC3 {EQS Score}_{zone} = frac{{EQS}_{A} + {EQS}_{B} + {EQS}_{C}}{3} 

  4. The resulting Zone EQS Score determines the EQS Category to be applied to each sampling zone, as shown in Table 10:

    Table 10. EQS Categories associated with the Zone EQS Scores.

    Zone EQS Score

    1.0

    1.1 to 2.0

    2.1 to 3.0

    3.1 to 4.0

    >4.0

    EQS Category

    High

    Good

    Moderate

    Poor

    Bad

  5. The EQS Category obtained from Table 10 shall be compared with Table 11 to determine the benthic status of the sampling zone. 

Table 11. EQS Categories for “Acceptable’ benthic status.

Sampling Location Zones

EQS Category for ‘Acceptable’ Benthic Status

Inside Site Boundary

At least ‘Moderate’ EQS Category shall be achieved

Outside Site Boundary

‘Poor’ or “Bad” EQS Category are not acceptable categories

7.5 Method for Freshwater Systems Discharging into Rivers (Indicator 2.5.4)

7.5.1 Sampling Method

  1. Sediment sampling for macrofauna taxonomic analysis shall be conducted yearly in the receiving river in at least three transects 10 metres apart,  upstream and downstream from the site outflow point. Each transect shall contain at least four sampling locations for a minimum of 24 samples.

    • The sampling shall focus on key sensitive Indicator species for the region.

    • The transects shall be located to include sampling locations midstream, near the bank and in areas with slower water flow.

    • The sampling locations shall include areas with similar conditions, such as bottom type, water flow and/or substrate types, along the bank in the upstream and downstream sampling locations.

    • The position of the sampling locations downstream shall reflect a scientific assessment of the most likely area of potential impact from the site, considering the mixing of water and the minimum and maximum distance from the site outflow point.

  2. The samples shall be analysed to determine the presence, composition, abundance and diversity of invertebrate benthic fauna.

  3. With the results of the analysis of the samples, the benthic status for the upstream and downstream sampling locations shall be determined using a categorisation system that includes at least five categories of benthic status.

  4. The determined category downstream shall be compared with the category upstream to demonstrate that the benthic status of the river does not deteriorate downstream from the site’s outflow point.

In case the downstream sampling locations drop in benthic status category, two sediment sampling for macrofauna taxonomic analysis shall be conducted during the next 12-month period following the method outlined in this section and using the previously used categorisation system.

After three years of demonstrating conformance with Indicator 2.5.4, sites may reduce sampling to once per certification cycle (i.e. every three years) as long as there have been no significant changes to farming practices and thus that previously determined benthic status provides a credible proxy for future performance.

7.6 Method for determining the distance between the site and seagrass habitats (Indicator 2.2.5)

  1. The zone 1 boundary of the site shall be identified, in accordance with Figure 1 outlined in Section 7.2.1.

  2. The nearest edge of seagrass habitat from the boundary of the site shall be determined by the Environmental Risk Assessment (Appendix 3 (3.3)).

  3. The distance to the nearest edge of seagrass habitat shall be then be identified from the zone 1 boundary.

7.7 Method for Cage Culture in Marine/Brackish Waters Using Copper Nets or Copper-Treated Nets (Indicator 2.11.11)

7.7.1 Sampling Method

  1. Sediment sampling shall be conducted in duplicate in three of the transects required by the Level 2 monitoring sampling method for Cages in Marine/Brackish Waters outlined in Section 7.2.1.

    • Of the three transects, one should be upstream and one downstream with respect to the direction of the predominant current.

  2. In each transect, samples shall be collected in Zone 2 and at the reference site outlined in Figure 1.

  3. The samples shall be analysed for copper levels expressed in mg Cu/kg dry sediment weight, and the result compared with the metric required by Indicator 2.11.11.

  4. Sampling shall be conducted at the same time as per the timing required by the benthic sampling method for cages in marine/brackish waters outlined in Section 7.2.1.

7.8 Methods for Field Analysis of Abiotic Indicators (Indicator 2.5.1)

7.8.1 Total Free Sulphide – UV Spectrophotometry (S-2UV)

The method is described and assessed in Cranford (2024)134134Cranford, P. J. (2024). A simple and rapid method for measuring total free sulfides in marine sediments. Limnology and Oceanography: Methods. https://doi.org/10.1002/lom3.10619 and is listed as a recognised, validated method in the US National Environmental Methods Index (listed as “A simple and rapid method for measuring total free sulfides in marine sediments135135National Environmental Methods Index”). This field method includes the extraction and analysis of porewater in surficial sediments collected by core or grab samplers.

Standard Operating Procedure

  1. Drain water in the sediment sampler to the sediment surface.

  2. Three Rhizon tubes are inserted into the sediment between the undisturbed surface and 2cm depth. Note that a 5cm long Rhizon is inserted at an angle to sample within the 0–2 cm depth range.

  3. Flush each tube by withdrawing 0.5–1.0mL of porewater into each syringe. Discard the flushing water inside the syringe.

  4. A 100-µL sample of porewater is immediately removed from inside each Rhizon tube by inserting the needle attached to a 100-µL gas-tight syringe. An extension tube or adapter may be necessary between the Rhizon and syringe to hold sufficient sample volume.

  5. The 100-µL sample is immediately added to a 1.4mL capacity semi-micro quartz cuvette (1cm pathlength) along with 1mL of deionised water having a pH of approximately 8. The pH of this alkaline dilution water is previously adjusted with ammonium hydroxide and confirmed with pH-Indicator strips.

  6. Immediately place the cuvette into a mobile UV spectrophotometer and measure light absorption at 230, 240 and 250nm wavelengths. The instrument is to be frequently blanked with a cuvette containing the alkaline dilution water.

  7. Calculate the total free sulfide concentration using the absorbance values and the regression equations determined by the calibration procedure below. Although absorption data are collected for three wavelengths, S2- is only calculated using the lowest wavelength that provides absorbance readings below 2. If the absorbance reading at 230nm is >2, then use the 240nm absorbance, etc.

UV Spectrophotometer Calibration

The calibration of the UV spectrophotometer is highly stable and only needs to be conducted once a month to ensure the instrument has not been damaged. Calibration of UV spectrophotometers ensures accurate and reliable measurements and involves assessments of wavelengths and absorbance accuracy and linearity. The following equations shall be used to calibrate total free sulphide (S2-) concentrations from absorbance data collected at the three wavelengths:

  • S2 -(µM) = absorbance230 nm x 1580

  • S2- (µM) = absorbance240 nm x 2388

  • S2- (µM) = absorbance250 nm x 6788

One of the following four methods shall be used to calibrate the UV Spectrophotometer:

Certified Reference Material (CRM)

A CRM or Reference Standard is used as the stock solution for preparing five working standards of known sulfide concentration between 0 and 15,000µM.

  1. Dilute the stock CRM solution to prepare five known concentrations using pipettors and the alkaline deionised water.

  2. Blank (zero) the instrument and then add 100µL of the working standard to the cuvette along with 1mL of alkaline water. Record the light absorbance of each working standard at 230, 240 and 250nm wavelengths.

  3. Calculate the calibration equations for each of the three wavelengths using regression analysis (x= absorbance at selected wavelength and y= working standard concentration in µM units) while excluding any absorbance readings above 2.

Sulphide Standard Method 4500-S2-A

  1. Prepare the standards from sodium sulphide nonahydrate (Na2S · 9H2O) crystals.

  2. Verify concentration of stock solution using the iodometric method (4500-S2-F).

Note that the standards are technically difficult to accurately prepare and are not certified. However, they can serve to assess wavelength accuracy (peak S2- absorbance at 230 nm) and measurement linearity.

Nicotinic acid reference for wavelength absorbance and linearity assessment

  1. Accurately weigh approximately 100 mg of nicotinic acid standard and dissolve it in 100 mL of 0.1 M hydrochloric acid to prepare a stock solution of 1 mg/mL. Dilute the stock solution with 0.1 M hydrochloric acid to prepare a series of working standards with concentrations in the desired linear range (e.g., 5-25 μg/mL).

    1. Sealed certified reference sets are commercially available.

  2. Use 0.1 M hydrochloric acid as the blank to zero the instrument when using in-house prepared standards.

    1. Nicotinic acid should give a spectral scan containing characteristic peaks at 213 nm and 261 nm.

  3. To evaluate linearity, plot a graph of concentration against absorbance for each of the two wavelengths. If using a commercial reference set, the absorbance scale is verified by assuring that the absorbance readings on your instrument are within the readings on the certificate supplied with the reference set.

Commercial accredited sources: Starna Cells, Sigma-Aldrich, Carl Roth, US Pharmacopeia

Holmium oxide reference for wavelength assessment

  1. Dissolve 4 % weight per volume high purity holmium oxide in 100 mL of 1.4 M perchloric acid filled into a stoppered 10 mm quartz cuvette.

  2. Note the four characteristic absorption peaks at 241.13, 249.79, 278.10, 287.18 nm.

    1. Acceptable wavelength accuracy tolerance is ±1 nm at each wavelength or certified spectrum by the manufacturer.

Commercial accredited sources: NIST-traceable reference materials from Starna Cells (UK), Hellma Analytics (Germany), Reagecon (Ireland), Camspec (UK), Thermo Scientific, Holmium glass solid reference.

Materials List

  • UV Spectrophotometer that accepts a 10 mm cuvette and includes the 230 to 250 nm wavelength range. If used on small boats, the instrument should be compact and have no moving parts. The instrument can be powered by internal batteries (for example, IMPLEN C40 mobile nanophotometer) or an external source such as a small power station.

    • Additional examples of suitable instruments include the DeNovix DS-C or DS-7, SpectraMax QuickDrop and Cole-Parmer SP-500-UV.

  • Semi-micro quartz cuvette, 200-2500 nm spectral range, 10 mm pathlength, 1.4 ml capacity. Helma Analytics cuvettes available from Sigma-Aldrich (Part No. Z600237 or Z600288)

  • 5cm MacroRhizon porewater samplers. Part No. 19.21.32 from Rhizosphere (https://www.rhizosphere.com/rhizons/macrorhizons/).

  • 10cc Luer Lock syringes for porewater extraction. Automatic extraction (applies constant vacuum) can be performed with a locking syringe (e.g. VacLok®. www.qosina.com) or by inserting a stainless-steel compression spring inside a plastic or glass BDTM syringe (for example, Qosina No. C3307).

  • 100µL gas-tight syringes (for example, Hamilton 1700 series gastight syringe Part No. 81085 or 81065 available from Sigma-Aldrich). It is recommended to rinse the syringe with fresh water at the end of each day to prevent the needle clogging with salt.

  • 1mL pipettor or bottle-top dispenser for rinsing cuvette for adding the alkaline water to the cuvette and for preparing standards.

  • Ammonia hydroxide, 0.44M or similar concentration.

  • pH Indicator papers for adjusting the dilution water to ~8 (For example, Whatman Indicator papers; 4.5 – 10.0 range – No. WHA2614991 from Sigma-Aldrich)

  • Lint-free optical wipes (e.g. Kimwipes) for cleaning cuvette surfaces.

  • Concentrated Sulfide ISO Certified Reference Material or Reference Standard. An example of a tested and calibrated material is Absolute Standards, Inc. Sulfide (S): No. ABS54139 from Chromatographic Specialties Inc.

7.8.2 Total Free Sulphide – Ion Selective Electrode (S-2ISE)

The following procedure is based on Standard Method 4500-S2--F (APHA 2017) for water samples and the Canadian Aquaculture Activities Regulations Guidance Document (www.dfo-mpo.gc.ca) for sediment samples.

Standard Operating Procedure

  1. Prior to field measurements, prepare the Sulfide Anti-Oxidant Buffer (SAOB). Dissolve 20.0g NaOH (sodium hydroxide) crystals in 250ml of de-aerated deionised water (use a volumetric type flask). Add 17.9g EDTA (ethylene diamine tetra acetic acid disodium salt dehydrate) to NaOH solution and swirl until dissolved. Place solution in plastic screw top jar. Larger volumes can be prepared as necessary provided ratio of reagents is maintained. Add L-ascorbic acid just prior to analysis of samples. Store in the dark at 4°C. De-aerated deionised water must be at a similar temperature as that of the sediment to be sampled. The SAOB expiration period is 3 hours.

  2. The Ag+/S= combination electrode must be recalibrated a minimum of once every 3 hours and each time sampling is initiated at a new site.

  3. The tip of the electrode should be gently cleaned using an abrasive strip or detergent solution before each calibration. If difficulty is experienced in obtaining the expected slope for the relationship between mV potential and log sulfide concentrations cleaning may be necessary to remove coatings formed on the electrode tip.

  4. The level of the electrode filling solution should be checked and either topped up or replaced with fresh electrolyte (if the latter option is chosen, wait at least 30 minutes for electrode to stabilise prior to using it).

  5. In order to measure S2–ISE a 1:1 volume ratio of SAOB to sediment is required. Place 10 mL of SAOB in a small plastic or glass graduated container and then add surface sediment (0 – 2cm depth) until the meniscus is at the 20mL gradation (always add SAOB first).

  6. Briefly stir the mixture and then insert the Ag+/S= electrode into the sample. Ensure the tip of the electrode is fully covered by the SAOB/sediment slurry. Gently move the electrode in the slurry until the electrode stabilises. Depending on the electrode, a ‘Ready’ message appears on the meter screen and/or a beep is heard (typically requiring 1 – 4 minutes to stabilise).

  7. Gently wipe the electrode and remove all sediment prior to insertion into the next sample.

  8. Any oily residue on the electrode must be removed prior to further use.

  9. If potentials recorded with the Ag+/S= electrode in a sediment sample do not stabilise in 1 – 2 minutes, performance can be assessed by determining the S2-ISE concentration in one of the working standards that has been stored in a dark cooled container with a minimum head space. Do not attempt to correct the data for drift if deviations are less than 10 – 20% of expected standard values. If measured values have a greater deviation than the expected value a full recalibration should be performed following the calibration protocols.

  10. Upon completion of sediment measurements rinse the electrode and drain filling solution (unless electrode will be used within week).

Sulfide (Ag+/S=) Electrode Calibration

Ag+/S= electrodes shall be calibrated before the start of each sampling event and recalibrated a minimum of every 3 hours during an analytical session.

The following protocols are generic to the Ag+/S= electrode. Specific models may have slightly different specific requirements and the operator must be familiar with the model prior to calibrating the electrode. If routinely used, Ag+/S= combination electrodes should be replaced every couple of years as they tend to become less accurate due to wear and tear.

S2- Calibration Solutions:

Stock Standard:

Working in a fume-hood or well-ventilated area, add 0.2402g Na2S•9H2O to a volumetric flask containing 100 mL of de-aerated deionised water. Swirl until dissolved. Store this 10,000µM stock standard in a refrigerated in dark, air-tight bottle with minimum head space. The stock solution expiration period is two days. Stock standards should be prepared strictly according to Standard Method 4500-S2- (APHA 2017), which provides information on the safe handling and storage of the stock crystals, and the verification of the prepared stock concentration using titration methods (e.g. Iodometric Standard Method 4500-S2--F).

Working Standards:

Prepare a serial dilution (10,000μM, 1,000μM, 100μM and 10μM (if required) of S2- standards immediately before calibrating the Ag+/S= combination electrode by transferring 10mL of the 10,000μM Na2S stock solution into a volumetric flask and diluting with 90mL of de-aerated, deionised water. This procedure is repeated sequentially using 10mL of each successive standard and diluting with 90mL of de-aerated, deionised water. The de-aerated, deionised water must be at a similar temperature as that of the sediment to be sampled. The working standard expiration period is 3 hours.

10,000μM, 1,000μM, 100μM three working standards are recommended for the 3-point calibration. If samples are expected or found to have ‘S’ concentrations below 100μM then a 10μM standard should be prepared to create a 3-point calibration series consisting of 10μM, 100μM and 1,000μM. ‘S’ solutions below 100μM are unstable and these values are also at the minimum sensitivity of most commercially available Ag+/S= combination electrodes.

Calibration Procedure:

  1. Calibration of the Ag+/S= combination electrode is to be carried out by working from the lowest to highest concentration in a standard 3-point series. A fixed volume of a standard is diluted with an equal volume of SAOB (1:1 by volume) immediately before each potential is measured.

  2. The range of standards used for the 3-point calibration should bracket the range of sulfide concentration expected in the sediment samples.

  3. mV potentials are stored by the multimeter after they become stable (usually between 1 – 3 minutes depending on the make of the meter).

  4. Upon completing the measurement of the third standard instruct the meter to calculate the slope of the concentrations. The theoretical slope is -28mV for each 10-fold increase in the ‘S’ concentration but because of temperature sensitivities and differences in electrodes the accepted range of values for the slope is between -27 to -33. If the slope is outside this range, repeat the calibration using fresh standards (also check the cleanliness of the probe, filling solution level, battery function, etc. prior to recalibrating).

  5. Following calibration, rinse the electrode with deionised water and blot dry before measuring the first sample.

Materials List

  • Ag+/S= combination electrode and appropriate filling solution.

  • Multimeter.

  • NaOH (sodium hydroxide) crystals.

  • EDTA (ethylene diamine tetra acetic acid disodium salt dehydrate).

  • L-ascorbic acid.

  • De-aerated, de-ionised water

  • Na2S•9H2O crystals.

  • Assorted glasswear and pipettors.

7.8.3 Redox Potential (Eh)

Eh can be measured directly in the grab/core using an Oxidation Reduction Potential (ORP) probe that uses a silver/silver chloride or platinum reference electrode. The ORP probe must be calibrated, operated and maintained according to strict manufacturer specifications. ORP measurements (referred to as ORP, EAg/AgCl or EPt), are by themselves ambiguous and it is only through specifying the reference scale that the data be interpreted. ORP measurements converted to a hydrogen scale are reported as ‘Eh’ and some publications designate the same measurements as EhNHE. ORP data (mV) obtained in the field with Ag/AgCl or Pt electrodes are converted to the hydrogen scale as follows:

Eh = ORP (mV) + half-cell potential of reference electrode,

Where the half-cell potential of the Ag/AgCl or Pt reference electrode is related to the molarity of the probe filling solution and measurement temperature (see Table 12).

Table 12 Half-cell potential of Ag/AgCl reference electrode.

T (°C)

Molarity of KCl (Potassium chloride) filling solution

1.5M

3M

3.3M

3.5M

4M

5

254

224

220

219

219

10

251

220

217

215

214

15

249

216

214

212

209

20

244

213

210

208

204

25

241

209

207

205

199

30

238

205

203

201

194

Standard Operating Procedure

  1. ORP readings are conducted at the same time as porewater is being extracted for S2-UV analysis.

  2. The ORP probe is inserted directly into the sediment surface inside the core/grab. Gently mix the surface sediment (0 and 2cm depth) around the probe location with the probe and leave the probe in the mixed sediment area. Ensure full contact between the ORP electrode tip and wet sediment.

  3. The ORP mV reading should stabilise within 1–2 minutes. If redox conditions are not controlled by single oxidation-reduction reactions, as in oxic sediments, there is often a slow, continuous drift of electrode potentials. An arbitrary time (3–4 minutes) can be chosen to record mV readings if they do not stabilise sooner. Potentials in reduced sediments usually stabilise more rapidly.

  4. Record the sediment sample temperature.

  5. Correct the ORP potential (mV) relative to the normal hydrogen electrode as described above using manufacturer information on the electrode filling solution and the recorded sediment temperature.

Materials List

  • A rugged Oxidation Reduction Potential probe (e.g. Hamilton Polyplast ORP BNC).

  • A compatible waterproof portable mV meter with temperature probe (e.g. Hanna HI9126).

  • ORP electrode storage solution.

  • ORP buffer solutions with known mV readings for instrument testing.

7.8.4 pH

As with the Eh measurements, pH is to be measured directly in the grab/core with an electrode. The pH can be measured either with an ordinary glass electrode or with an Ion Specific Field Effect Transistor (ISFET) based device. The ISFET sensors are very robust in comparison with glass electrodes and are available with a conic tip that can be inserted into the sediment. The pH electrode potentials are temperature dependent, so the pH meter must be equipped with temperature compensation.

  1. The pH electrode is set up and calibrated according to the manufacturer instructions. A two-step calibration of the electrode is carried out on the site before measurements begin using two buffers with a pH of 7.0 and 4.0.

  2. The electrode is ready for use when it gives stable readings when placed in a container with freshwater or a buffer. Stir occasionally.

  3. The pH electrode is inserted to a depth of 1cm into the sediment and kept as stable as possible. The pH value is read and recorded when the value is stable.

  4. Between samples, the electrode is rinsed and kept in a buffer with the same temperature as the samples.

  5. Regular checking of the pH of the buffer allows the electrode accuracy to be monitored. Discrepancies larger than 0.2 pH units from the first measured value require recalibrating the electrode.

Materials List

  • Field pH meter.

  • pH electrode (glass combination electrode set or ISFET electrode).

  • Buffers with pH 4.0 and pH 7.0 and distilled water.

7.8.5 Total Ammonia Nitrogen (TAN)

Hach® Company gained USEPA Equivalence on a simple salicylate method for use in wastewater based on the TNTplus™ Ammonia Nitrogen platform. This is a simple, cost-effective, 15-minute test, requiring no calibration and 0.5mL of porewater.

Standard Operating Procedure

  1. Extract porewater directly inside the grab sample as described in Section 7.8.1 for Total Free Sulphide – UV Spectrophotometry (S-2UV).

  2. Analyse the sample as soon as possible. During TAN analysis, the pH of the porewater sample must be between 4 and 8, and the temperature of the sample and reagents must be between 20–23°C.

  3. Pipette 0.5mL of sample into the TNT 831 DosiCap vial and, with the cap on, shake vigorously. Warm the sample in a water bath if necessary.

  4. After 15 minutes, clean the outside of the vial and insert it into the reader. Select the TNT 831 method for the spectrophotometer and press ‘READ’.

  5. The test procedure is to be evaluated monthly using a 10mg/L NH3-N Wastewater Effluent Ammonia Nitrogen Standard Solution.

Materials List

  • Hach DR1900 portable spectrophotometer.

  • Hach TNTplus 831 Low Range (1-12 mg/L NH3-N) reagent kits, which each contain 25 test vials.

  • Hach 10 mg/L NH3-N Wastewater Effluent Ammonia Nitrogen Standard Solution.

  • Pipette and tips.

7.8.5.1 TAN – Determination of the Moderate EQS Category Threshold

TAN consists of ammonium ion (NH4+) and un-ionised ammonia (NH3). NH3 makes up a higher proportion of TAN at higher pH and is typically associated with most of its toxic effects. Given the effects of temperature and pH on the speciation and toxicity of TAN, the Moderate EQS threshold from Table 1 (1.9 mg/L) only applies to sediment sample conditions of pH 7 and 20°C. The following formula shall be used to determine the Moderate EQS threshold to be applied to other pH and temperature sediment sample conditions:

 TAN Moderate EQS threshold(mgL)=0.8876×(0.02781+107.688−pH+1.19941+10pH−7.688)×(2.126×100.028×(20−T)) TAN Moderate EQS threshold left( frac{mg}{L} right) = 0.8876 times left( frac{0.0278}{1 + 10^{7.688 – pH}} + frac{1.1994}{1 + 10^{pH – 7.688}} right) times (2.126 times 10^{0.028 times (20 – T)}) 

Where the TAN Moderate EQS threshold is calculated for each sampling zone using the average temperature (T, in˚C) and pH measurements of the sampling zone.