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Reference
Endpoint:
sediment toxicity: long-term
Type of information:
experimental study
Adequacy of study:
key study
Study period:
May 2016 - February 2017
Reliability:
1 (reliable without restriction)
Justification for type of information:
The data from this study is required by the European Food Safety Authority (EFSA) who recommend the testing of three marine species to assess the safety of feed additives to the environment.
Qualifier:
according to
Guideline:
other: guidance from OSPAR 2005
Deviations:
yes
Remarks:
With modifications for a 28 day test using juvenile Corophium based on study designs published in the open scientific literature
Principles of method if other than guideline:
This test method is using guidance from OSPAR 2005. (Protocols on Methods for the Testing of Chemicals Used in the Offshore Oil Industry Part A - A Sediment Bioassay using an Amphipod Corophium sp.), With modifications for a 28 day test using juvenile Corophium based on study designs published in the open scientific literature. As there are no internationally agreed guidelines for chronic Corophium tests at this time it has not undergone rigorous inter-laboratory comparison, however, this is deemed the most appropriate test design for the purpose of the product assessment by EFSA. The study was conducted to GLP standards at CEFAS Lowestoft laboratory.
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
Analytical samples were taken on days 0, 7 and upon test termination (day 28), from each test concentration. Day 0 replicates contained no corophium, whereas day 7 and 28 replicates contained 30 individuals per replicate. Once collected, samples were stored frozen until analysis. The concentration of GLDA-Na4 in samples was determined using ultraperformance liquid chromatography with ultraviolet detection (TUV) Refs 12-14.
Vehicle:
no
Details on sediment and application:
Combined water and sediment collected from Lowestoft Bay
Test organisms (species):
Corophium volutator
Details on test organisms:
The test organism was Corophium volutator and juveniles ≤ 7 days old were used for the test. Gravid females supplied by: Ecospan Unit 8 Strashleigh View, Lee Mill Industrial Estate, Plymouth, Devon, PL21 9GS. The gravid females were cultured in the laboratory until enough juveniles were produced for the test.
Study type:
laboratory study
Test type:
semi-static
Water media type:
saltwater
Type of sediment:
natural sediment
Remarks:
No details on sediment
Limit test:
no
Duration:
28 d
Exposure phase:
other: juvenile (≤ 7 day old) estuarine amphipods (Corophium volutator)
Test temperature:
14.7 - 15.9 (one day a temperature of 17.2 was observed due to an airco failure)
pH:
7.7 - 8.2
Dissolved oxygen:
≥60%
Salinity:
30 ± 3‰
Nominal and measured concentrations:
Nominal concentrations (mg/kg active ingredient): 0, 460, 1000, 2200, 4600, 10000


A solubility test was carried out using approximately 1 g of Dissolvine GL-47-S to 1 L of natural seawater (collected from Lowestoft bay and filtered to 0.2 µm). The solution was stirred for 2 hours and observed to determine solubility. Dissolvine GL-47-S was found to be soluble in seawater, therefore procedure A in SOP 2114 could be used.
Reference substance (positive control):
no
Duration:
28 d
Dose descriptor:
EC50
Effect conc.:
1 144 mg/kg sediment dw
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
mortality
Key result
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
413 mg/kg sediment dw
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
mortality
Duration:
28 d
Dose descriptor:
EC50
Effect conc.:
> 1 984 mg/kg sediment dw
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
other: Length
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
< 197.8 mg/kg sediment dw
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
other: Length
Duration:
28 d
Dose descriptor:
EC50
Effect conc.:
1 436 mg/kg sediment dw
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
biomass
Key result
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
412.4 mg/kg sediment dw
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
biomass
Details on results:
Mortality:
For Mortality using the day 0 actuals, the median lethal concentration (EC50) was 3010 mg/kg with 95% confidence limits between 2803 - 3233 mg/kg. Using the day 28 actuals, the median lethal concentration (EC50) was 434.5 mg/kg with 95% confidence limits between 404.6 – 466.5 mg/kg. The EC50 geometric mean was 1143.61 mg/kg. An EC10 could not be calculated using the spearman karber model.

The day 0 actuals No Observed Effect Concentration (NOEC) for mortality was 1097 mg/kg and the Lowest Observed Effect Concentration (LOEC) was 3231 mg/kg. For day 28 actuals, the NOEC for mortality was 155 mg/kg and the LOEC was 476 mg/kg. The NOEC geometric mean for mortality was 412.35 mg/kg and the LOEC was 1240.14 mg/kg.

Length per animal (mm):
For Length using the day 0 actuals, the EC50 was >5270 mg/kg and the EC10 was 471.1mg/kg, with 95% confidence limits between 0 – 1874 mg/kg. Using the day 28 actuals, the EC50 was >747 mg/kg. The 28 day actuals EC10 for length was 61.04 mg/kg with 95% confidence limits between 0 – 282.6 mg/kg over 28 days. The EC50 geometric mean for length was 1984.11 mg/kg, and EC10 geometric mean for length was 169.57 mg/kg.

The day 0 actuals NOEC for length was <505 mg/kg and the LOEC was 505 mg/kg. For day 28 actuals, the NOEC for length was <64 mg/kg and the LOEC was 64 mg/kg. The NOEC geometric mean for length was <179.78 mg/kg, and LOEC geometric mean for length was 179.78 mg/kg.

Weight per animal (g):
For weight using the day 0 actuals, the EC50 was 3700 mg/kg with 95% confidence limits between 2047 - 6687 mg/kg. The EC10 was 449 mg/kg, with 95% confidence limits between 8 - 968 mg/kg. Using the day 28 actuals, the EC50 was 557 mg/kg with 95% confidence limits between 293 - 1058 mg/kg. The 28 day actuals EC10 for weight was 60 mg/kg, with 95% confidence limits between 0 – 137mg/kg. The EC50 geometric mean for weight was 1435.58 mg/kg, and EC10 geometric mean for weight was 164.13 mg/kg.

The day 0 actuals NOEC for weight was 1097 mg/kg and the LOEC was 3231 mg/kg. For day 28 actuals, the NOEC for weight was 155 mg/kg and the LOEC was 476 mg/kg. The NOEC geometric mean for weight was 412.35 mg/kg, and LOEC geometric mean for weight was 1240.14mg/kg.
Reported statistics and error estimates:
Statistical methods

The data were analysed using Comprehensive Environmental Toxicity Information System (CETIS) statistical software (Tidepool scientific, V1.8.0.9) and following CEFAS SOP 2100: “The use of the statistical software CETIS.” Refs 10-11.
The EC50 and the EC10 for survival, weight and length were calculated using Linear interpolation, Spearman Karber or Non-linear regression log-logistic models and the NOEC and LOECs were calculated using Dunnetts Multiple comparison test or Bonferroni Adjusted T-tests.
Graphs for mortality, length and weight were created in Graphpad prism 6 (version 6.07, Graphpad software inc ©). Graphs of statistical models were created in CETIS.

Chemistry Analysis

The percentage recovery of GLDA-Na4 (Dissolvine GL-47-S active ingredient) was 100 – 151% of nominals, for water and sediment samples combined, for day 0 and day 7. (see Annex 3 for all analytical phase results). By day 28 the recovery of GLDA-Na4 was 14 – 22 % of nominals, for water and sediment samples combined. The concentration reduction was likely due to the overlying water renewals because GLDA-Na4 partitions into the water from the sediment. This was shown in the day 0 to day 7 results where 71 – 108 % of GLDA-Na4 was found in the water across the concentration range. Therefore, some of the test item would have been removed from the test system when the overlying water was renewed with clean seawater. The chemistry results are summarised in tables 4 to 6.

 

Table 4: Day 0 chemistry analysis results

 Day 0

 

 

 

Nominal GLDA
(mg/kg)

mg recovered from sample (total)

% recovery

GLDA Actuals*
(mg/kg)

460

261

110

505

1000

567

110

1097

2200

1670

147

3231

4600

2724

115

5270

10000

5845

113

11308

*Actuals based on average dry weight of sediment in the test vessel being 0.517 kg

 

Table 5: Day 7 chemistry analysis results

 Day 7

 

 

 

Nominal GLDA
(mg/kg)

mg recovered from sample (total)

% recovery

GLDA Actuals*
(mg/kg)

460

239

100

462

1000

531

103

1027

2200

1715

151

3318

4600

2758

116

5336

10000

5707

110

11041

*Actuals based on average dry weight of sediment in the test vessel being 0.517 kg

 

Table 6: Day 28 chemistry analysis results.

Day 28 

 

 

 

Nominal GLDA
(mg/kg)

mg recovered from sample (total)

% recovery

GLDA Actuals*
(mg/kg)

460

33

14

64

1000

80

16

155

2200

246

22

476

4600

386

16

747

10000

749

14

1449

*Actuals based on average dry weight of sediment in the test vessel being 0.517 kg

All reported results are based on day 0 and day 28 actuals for GLDA concentration.

 

Toxicity results (Day 0 and Day 28 actuals)

Table 7: P0088L summary of toxicity of Dissolvine GL-47-S to Corophium volutator, using day 0 and day 28 actual active ingredient (GLDA -Na4) chemistry results.

Corophium 28-day Study Results

Geometric mean

GLDA – Na4 mg/kg

Day 0 measured results

Day 28 measured results

Statistical methods

GLDA-Na4

mg/kg

95% Confidence limits

GLDA-Na4mg/kg

95% Confidence limits

Mortality EC50

 

1144

3010

2803 - 3233

434.5

404.6 - 466.5

Spearman Karber

Mortality NOEC

 

413

1097

-

155

-

Dunnetts Multiple comparison test, Bonferroni Adjusted T-test

Mortality LOEC

 

1240

3231

-

476

-

Length EC50

 

>1984

>5270

-

>747

-

Linear interpolation

Length EC10

 

169.6

471.1

0 - 1874

61.04

0 – 282.6

Length NOEC

 

<179.8

<505

-

<64

-

Bonferroni Adjusted T-test

Length LOEC

 

179.8

505

-

64

-

Weight EC50

 

1436

3700

2047 - 6687

557

293 - 1058

Non-linear regression log-logistic model

Weight EC10

 

164.1

449.3

8 - 968

60

0 - 137

Weight NOEC

 

412.4

1097

-

155

-

Dunnetts Multiple comparison test

Weight LOEC

 

1240

3231

-

476

-

Physico-chemical properties of test solutions

Table 6: Water Quality ranges for the test solutions during the definitive study P0088L.

 

Concentration (mg/kg)

Actuals Day 0 (day28)

pH

Salinity (ppt)

D.O (%ASV)

Temperature (°C)

Control

Min

7.8

29.3

87.3

15.0

Max

8.1

31.9

101.5

15.9

505 (64)

 

Min

7.7

29.3

97.8

15.1

Max

8.1

31.6

101.3

15.6

1097 (155)

 

Min

7.9

29.5

92.5

15.0

Max

8.1

32.2

101.3

15.7

3231 (476)

 

Min

7.9

29.7

90.6

14.8

Max

8.2

32.1

101.2

15.5

5270 (747)

 

Min

8.0

30.0

94.4

14.9

Max

8.1

32.1

100.9

17.2*

11308 (1449)

 

Min

8.0

30.1

90.9

14.7

Max

8.2

32.7

101.1

15.6

*see deviations Annex 1

Validity criteria fulfilled:
yes
Conclusions:
The test is considered valid as all validity criteria have been fulfilled also considering the test setup used. (GLP, Guideline study with adaptations)
Executive summary:

The long term toxicity of L-glutamic acid, N,N-diacetic acid, tetrasodium salt (GLDA-Na4) to the benthic amphipod Corophium volutator was assessed using the test guidance from OSPAR 2005. (Protocols on Methods for the Testing of Chemicals Used in the Offshore Oil Industry Part A - A Sediment Bioassay using an Amphipod Corophium sp.). This guidance was used with modifications for a 28 day test using juvenile Corophium as there are no internationally agreed guidelines for chronic Corophium tests at this time. Although it has not undergone rigorous inter-laboratory comparison this approach is deemed the most appropriate test design and uses study designs published in the open scientific literature, for the purpose of the product assessment by EFSA. The study was conducted to GLP standards at CEFAS Lowestoft laboratory.

 

In this study juvenile (≤ 7 day old) estuarine amphipods (Corophium volutator) were exposed to a range of concentrations of an aqueous solution of L-glutamic acid, N,N-diacetic acid, tetrasodium salt GLDA-Na4 (Dissolvine GL-47-S containing 47.5% GLDA-Na4) in sediment, with overlying seawater (30 ± 3‰) under semi-static test conditions, at 15 ±2˚C. Animals were exposed concurrently in untreated sediment which served as the control. After 28 days, the test replicates were sieved and the following endpoints were determined: the combined wet weight of the amphipods per replicate (to the nearest 0.1 mg) and all animals (≥3.00 mm), individually measured (to the nearest 0.1 mm). NOEC, LOEC and EC50 values for mortality and growth were calculated and are presented in the table below.

 

Results:         

Table: Results summary

Corophium 28-day Study Results

Geometric mean

GLDA – Na4 mg/kg

Day 0 measured results

Day 28 measured results

Statistical methods

GLDA-Na4

mg/kg

95% Confidence limits

GLDA-Na4mg/kg

95% Confidence limits

Mortality EC50

 

1144

3010

2803 - 3233

434.5

404.6 - 466.5

Spearman Karber

Mortality NOEC

 

413

1097

-

155

-

Dunnetts Multiple comparison test, Bonferroni Adjusted T-test

Mortality LOEC

 

1240

3231

-

476

-

Length EC50

 

>1984

>5270

-

>747

-

Linear interpolation

Length EC10

 

169.6

471.1

0 - 1874

61.04

0 – 282.6

Length NOEC

 

<179.8

<505

-

<64

-

Bonferroni Adjusted T-test

Length LOEC

 

179.8

505

-

64

-

Weight EC50

 

1436

3700

2047 - 6687

557

293 - 1058

Non-linear regression log-logistic model

Weight EC10

 

164.1

449.3

8 - 968

60

0 - 137

Weight NOEC

 

412.4

1097

-

155

-

Dunnetts Multiple comparison test

Weight LOEC

 

1240

3231

-

476

-

 

Description of key information

There are two sediment studies performed with L-glutamic acid, N,N-diacetic acid, tetrasodium salt (GLDA-Na4). One long term study with Corophium volutator and an acute toxicity study with Arenicola marina.

The study with the polychaete Arenicola marina is a 10 day acute whole sediment bioassay. Arenicola marina is a direct deposit feeder that is widely distributed in European coastal waters and on the east coast of North America and is recognised as a suitable test species for use in whole sediment reworker bioassays for toxicity testing.

The median lethal concentration (EC50) for survival was 3507 mg/kg of GLDA-Na4 (active ingredient) with 95% confidence limits between 3377 and 3569 mg/kg (sediment and water combined) over 10 days. The EC50 determined using sediment only concentrations of GLDA-Na4 was 983.2 with 95% confidence limits between 967.2 and 998.6 mg/kg dw over the same test period. It should be noted that GLDA-Na4 does not sorb to sediment and that the organisms were therefore only exposed via the aquatic compartment (porewater) and not via ingestion. Based on these facts the endpoint based on sediment and water combined should be used for risk assessment purposes. The EC50 for number of casts produced was 3509 mg/kg with 95% confidence limits between 1621 and 4232 mg/kg of GLDA-Na4 for combined water and sediment concentrations.

The long term toxicity study with the benthic amphipod Corophium volutator was performed using the test guidance from OSPAR 2005. This guidance was used with modifications for a 28 day test using juvenile Corophium as there are no internationally agreed guidelines for chronic Corophium tests at this time.

In this study juvenile (≤ 7 day old) estuarine amphipods (Corophium volutator) were exposed to a range of concentrations of an aqueous solution of L-glutamic acid, N,N-diacetic acid, tetrasodium salt GLDA-Na4 in sediment, with overlying seawater (30 ± 3‰) under semi-static test conditions, at 15 ±2˚C. Animals were exposed concurrently in untreated sediment which served as the control. After 28 days, the test replicates were sieved and the following endpoints were determined: NOEC, LOEC and EC50 values for mortality and growth

For Mortality the EC50 based on geometric mean measured concentration was 1143.61 mg/kg dw (active ingredient). A NOEC based geometric mean measured concentration was 412.4 mg/kg dw. For biomass an EC50 and NOEC were derived of 1436 and 412.4 mg/kg dw (active ingredient) respectively.

Key value for chemical safety assessment

EC50 or LC50 for marine water sediment:
1 143.6 mg/kg sediment dw
EC10, LC10 or NOEC for marine water sediment:
412.4 mg/kg sediment dw

Additional information