Tetrasodium N,N-bis(carboxylatomethyl)-L-glutamate

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July - November 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Justification for type of information:

The data from this study is required by the European Food Safety Authority (EFSA)

Qualifier:

equivalent or similar to guideline

Guideline:

other: This test method used guidance from ISO 14669 (1999) Water Quality - Determination of acute lethal toxicity to marine copepods (Copepoda, Crustacea) with modifications for a 21-day reproduction test allowing control nauplii to develop into adulthood.

Deviations:

not specified

Principles of method if other than guideline:

This test method used guidance from ISO 14669 (1999) Water Quality - Determination of acute lethal toxicity to marine copepods (Copepoda, Crustacea) with modifications for a 21-day reproduction test allowing control nauplii to develop into adulthood. As there are no internationally agreed guidelines for chronic Tisbe tests at this time it has not undergone rigorous inter-laboratory comparison, however, it was 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 the time points indicated below. Samples were stored frozen until analysis.
ON samples (pre renewal) OFF samples (post renewal)
12/09/16 07/09/16
14/09/16 14/09/16
16/09/16 19/09/16

Vehicle:

no

Details on test solutions:

As Dissolvine GL-47-S is known to be soluble in seawater, the test item stock was prepared in dilution water in a glass volumetric flask.
On the first day of the test, a small amount of dilution water (filtered, salinity adjusted and aged, seawater) was added to a 250 mL glass volumetric flask and the Dissolvine GL-47-S was weighed into a glass weigh boat and added to the volumetric flask, the pH was adjusted to be within acceptable limits prior to the volumetric flask being topped up to volume with dilution water. Aliquots of the stock solution were then taken and added to each 250 mL glass volumetric flask containing a small amount of dilution water as well as an appropriate volume of Tetraselmis suecica and Diacronema lutheri (to ensure test organisms had sufficient food within renewal days) before being topped up to volume with dilution water to make the test concentrations. This process was repeated on each subsequent renewal day with an increase in test concentration volumes to 500 mL where analytical samples were required to be taken.
Test solution renewals and recording of ON and OFF water qualities was conducted every Monday, Wednesday and Friday for the duration of the test.

Test organisms (species):

other aquatic crustacea: Tisbe battagliai

Details on test organisms:

The test organism was Tisbe battagliai, <24 hours old. Adult Tisbe battagliai were obtained from Guernsey Sea Farms Ltd (Parc Lane, Vale, Guernsey GY3 5EQ), acclimated to test conditions and cultured in the laboratory prior to initiation of the test. Females with egg sacks were isolated from the culture within 24 hours of the definitive test. This ensured that any nauplii present on Day 0 were <24 hours old. Upon test initiation the culture containing the females with egg sacks was gently poured through a 100 μm and then a 60 μm sieve. Adult Tisbe were collected on the 100 μm sieve and nauplii on the 60 μm sieve, nauplii were then
transferred to a holding tank containing control water. The nauplii were gently transferred from the holding tank into test vessels using glass pipettes.

Test type:

semi-static

Water media type:

saltwater

Limit test:

no

Total exposure duration:

21 d

Test temperature:

Temperature range: 19.7 – 20.7°C

pH:

pH range: 7.7 – 8.4

Dissolved oxygen:

Dissolved oxygen range: 96.8 – 106.1% ASV

Salinity:

Salinity range: 30.0 – 33.1‰

Nominal and measured concentrations:

The nominal test concentration ranged of 46 to 1000 mg/L of GLDA-Na4 (Dissolvine GL-47-S active ingredient).
The actual measured (average) concentrations for the definitive test were control (0), 39, 78, 224, 449 and 1056 mg/L GLDA-Na4 (Dissolvine GL-47-S active ingredient).

Details on test conditions:

The non-GLP range finding test was conducted with 20 replicates per concentration (one individual per replicate) with a nominal test concentration range of 46 to 1000 mg/L of GLDA-Na4 (Dissolvine GL-47-S active ingredient).
The duration of the non-GLP range finding test was 5 days. From the results of this, a definitive test concentration range was chosen. The definitive test was conducted with 40 replicates per concentration and 80 replicates for the control for the development phase and up to 20 replicates per concentration with 40 replicates for the controls for the reproduction phase depending on the ratio of males: females and survival rate by pairing day.
On the first day of the test, a small amount of dilution water (filtered, salinity adjusted and aged, seawater) was added to a 250 mL glass volumetric flask and the Dissolvine GL-47-S was weighed into a glass weigh boat and added to the volumetric flask, the pH was adjusted to be within acceptable limits prior to the volumetric flask being topped up to volume with dilution water. Aliquots of the stock solution were then taken and added to each 250 mL glass volumetric flask containing a small amount of dilution water as well as an appropriate volume of Tetraselmis suecica and Diacronema lutheri (to ensure test organisms had sufficient food
within renewal days) before being topped up to volume with dilution water to make the test concentrations. This process was repeated on each subsequent renewal day with an increase in test concentration volumes to 500 mL where analytical samples were required to be taken.
On day 0 T. battagliai nauplii <24 hours old were randomly selected and added individually to each test vessel to provide one animal per replicate, prior to addition of 2 ml of test solution or control water as appropriate, to the opposite side of the well using a pipette to avoid damaging the nauplii. Surplus test solution was also stored in analytical test well-plates as well as 150 mL beakers without T. battagliai, but stored under the same conditions as biological test vessels in order to collect analytical samples and record water quality parameters. Water qualities were recorded on pre renewal (ON) and post renewal (OFF) solutions. The tests were conducted in a controlled temperature room at 20 ± 2 °C monitored continuously, with a light:dark cycle of 16:8 hours. The experiment started once the test organisms (T. battagliai)
were added to the test vessels.
Under normal conditions T. battagliai develop into adults within approximately 10 days, therefore on day 12 adult males and females were taken from the 24-well plates and paired in 12-well plates for each test concentration and the controls with 5 mL of test solution per well.
Surplus males and females were also transferred to 12-well plates and their locations recorded, this allowed for movement of individuals within test concentrations where mortalities or suspected same-sex pairings occurred, movement of individuals was not conducted past day 17. Observations of mortality were made every day. Test solution renewals and recording of ON and OFF water qualities was conducted every Monday, Wednesday and Friday for the duration of the test. Following the day 12 pairing the presence of brood sacks in females and numbers of nauplii in wells was recorded. The test was terminated after 21 days.

Reference substance (positive control):

no

Key result

Duration:

21 d

Dose descriptor:

EC50

Remarks:

survival

Effect conc.:

396.2 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

act. ingr.

Basis for effect:

mortality

Remarks on result:

other: 95% conf lim: 322.3 - 486.9

Key result

Duration:

21 d

Dose descriptor:

NOEC

Remarks:

Number of Nauplii

Effect conc.:

224 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

act. ingr.

Basis for effect:

reproduction

Key result

Duration:

21 d

Dose descriptor:

EC50

Remarks:

number of Nauplii

Effect conc.:

273.7 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

act. ingr.

Basis for effect:

reproduction

Remarks on result:

other: 95% conf. lim: 153.2 - 334.4

Results with reference substance (positive control):

n.a.

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.”
Due to the nature of the data the survival EC50 was calculated using the Trimmed Spearman-kärber test and EC10 was calculated using Linear interpolation. The survival NOEC and LOEC were calculated using Fishers exact test.
Both the number of broods and number of nauplii EC50s were calculated using Linear interpolation and the NOEC and LOECs were calculated using the Wilcoxon/Bonferroni adjusted test.

Table 1: Definitive study 3 results P0092L

 

Tisbe 21-day Study Results	Actual measured (average) active ingredient conc. mg/L	95% Confidence limits	Statistical methods
Survival EC50	396.2	322.3 – 486.9	Trimmed Spearman Karber test
Survival EC10	87.74	8.680 – 235.9	Linear Interpolation
Survival NOEC	78	-	Fishers exact test
Survival LOEC	224	-	Fishers exact test
No. of Broods EC50	214.2	112.6 – 297.6	Linear Interpolation
No. of Broods NOEC	78	-	Wilcoxon/Bonferroni Adjusted test
No. of Broods LOEC	224	-	Wilcoxon/Bonferroni Adjusted test
No. of Nauplii EC50	273.7	153.2 – 334.4	Linear Interpolation
No. of Nauplii NOEC	224	-	Wilcoxon/Bonferroni Adjusted test
No. of Nauplii LOEC	499	-	Wilcoxon/Bonferroni Adjusted test

 

Non-GLP Rangefinder

Table 4: Dissolvine GL-47-S Range finding study % mortality and development results

Nominal Dissolvine GL-47-S Concentration (mg/L)	Nominal GLDA-Na4 Concentration (mg/L)	Mortalities	Percentage Mortality (%)	% Developed to Copepodid stage at day 5
Control (0)	Control (0)	1	5	30
97	46	1	5	45
211	100	0	0	40
463	220	1	5	30
968	460	0	0	20
2105	1000	0	0	0

 

Definitive (Tests 1 and 2)

 

These tests failed to meet the criteria for mortality in the control replicates and the data has not been used in this report.

 

Definitive(Test 3)

 

Table 5: P0092L Dissolvine GL-47-S Definitive test % mortality and reproduction results

 

Actual measured (average) GLDA-Na4 Concentration (mg/L)	Mortalities	Percentage Mortality(%)	Percentage of paired adults that produced ≥2 broods(%)	Average number of broods	Average number of Nauplii
0	3	4	82	2.3	61
39	8	20	69	1.6	53
78	2	5	91	2.0	53
224	8	20	36	1.1	40
499	25	63	0	0	0
1056	39	98	0	0	0

 

  Physico-chemical properties of test solutions

Table 7: Water Quality ranges for the test solutions during the definitive study 3, P0092L

 

Actual (average) GLDA-Na4 Concentration (mg/L)	Temperature (°C)	pH	Salinity (ppt)	D.O (%ASV)
0	19.9 – 20.9	7.7 – 8.3	30.0 – 33.0	96.8 – 104.7
39	19.9 – 20.7	8.1 – 8.4	30.0 – 33.1	99.4 – 106.1
78	19.8 – 20.6	8.1 – 8.4	30.0 – 32.9	99.3 – 105.5
224	19.9 – 20.7	8.1 – 8.4	30.0 – 32.9	99.6 – 105.5
499	19.9 – 20.6	8.2 – 8.4	30.0 – 32.9	100.1 – 105.3
1056	19.7 – 20.6	8.1 – 8.4	30.1 – 33.1	100.3 – 105.3

 

Validity criteria fulfilled:

yes

Conclusions:

Valid study performed under GLP conditions with chemical analysis.

Executive summary:

This study suggests that Dissolvine GL-47-S (containing 47.5% GLDA-Na4) causes toxicity, within the range of the chosen test concentrations, toTisbe battagliai over a 21-day test period.

 

For survival the median lethal concentration (EC₅₀) was 396.2 mg/L of GLDA-Na4 (Dissolvine GL-47-S active ingredient) with 95% confidence limits between 322.3 to 486.9 mg/L over 21 days. The 21-day No Observed Effect Concentration (NOEC) for survival was 78 mg/L of GLDA-Na4 (Dissolvine GL-47-S active ingredient) and Lowest Observed Effect Concentration (LOEC) was 224 mg/L of GLDA-Na4.

 

As well as assessing survival at 21 days the number of broods and number of nauplii was also assessed; the EC₅₀ for number of broods was 214.2 mg/L with 95% confidence limits between 112.6 to 297.6 mg/L of GLDA-Na4 (Dissolvine GL-47-S active ingredient), the NOEC and LOEC for number of broods were 78 mg/L and 224 mg/L of GLDA-Na4 respectively. The EC₅₀ for number of nauplii was 273.7 mg/L with 95% confidence limits between 153.2 to 334.4 mg/L of GLDA-Na4 (Dissolvine GL-47-S active ingredient), the NOEC and LOEC for number of nauplii were 224 mg/L and 499 mg/L ofGLDA-Na4 respectively.

 

From the dataset the EC₁₀for the survival endpoint could also be derived and is included in this report as extra information. The survival EC₁₀is as follows; 87.74 mg/L (95% confidence limits 8.680 – 235.9 mg/L).

Reference 2

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1 August 2008 - 2 February 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study according to international guideline (OECD guideline 211, EU guideline C.20) under GLP.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.20 (Daphnia magna Reproduction Test)

Deviations:

no

Principles of method if other than guideline:

Not relevant

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
Not relevant

Analytical monitoring:

yes

Details on sampling:

- Concentrations: nominal 0, 5, 13.5, 36.5, 98.4 and 265.7 mg/L
- Sampling method: At the start of the test, 0.75 mL was sampled from every test concentration including the control and directly put in a 2 mL HPLC vial. At test concentration renewal (fresh) 10 mL was was sampled directly from the freshly prepared test solution, before adding the daphnia's and algae. Sampling just before changing the test solutions (used) was done by taking 1 ml from every replicate (using a pipette) and pooling them in a glass sample bottle.
- Sample storage conditions before analysis: Samples taken at the start of the test were analyzed immediately. All other samples taken during the test were stored in the freezer until analyses.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION

Preparation of the stock solutions: To prepare the stock solutions for every water renewal, on average 0.2654 g (standard deviation: 0.0001 g) of test substance was weighed on an analytical balance then dissolved directly in 1L M4 (see § 3.7). The stock solutions were agitated mechanically for approximately 1 hour in an attempt to completely dissolve the test substance. The pH of each stock solution was checked and found to be between 7.8 and 8.3 and was therefore not adjusted. A fresh stock solution was prepared for each solution change.

Preparation of the test solutions: Test solutions were prepared by further dilution of the stock solution with M4. A geometric series of concentrations were used. The ratio between two consecutive concentrations was approximately 2.7. Test vessels were filled directly from the test solution containers immediately after preparation. The solutions were renewed three times a week during the test. The pH of the test solutions were all between 6 and 9 throughout the test and close to the value of M4 reconstituted water after each solution change. One control containing only test medium was included in the test.

Test medium: Reconstituted water (M4) according to Elendt (1990) (9.3) was used for culturing and in the test. The pH of this solution was 8.0 ± 0.5 and water hardness was above 140 mg/L (as CaCOs). The dilution water was aerated before being used in the test. The air was water-saturated and purified by activated charcoal and a cotton filter.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Strain/clone: clone 5
- Source: Dr. U. Noack-Laboratorien Käthe-Paulus- Str. 1, D-31157 Sarstedt, Germany)
- Age of parental stock (mean and range, SD): 2-4 weeks
- Feeding during test
- Food type: algal strain Chlorella vulgaris
- Amount: 0.2 mg carbon/daphnid/day

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Post exposure observation period:

Not relevant

Hardness:

> 140 mg/L CaCO3 (78.9-85.7 mg/L Ca2+ in control, 60.1-72.1 mg/L Ca2+ at test concentration 265.7 mg/L)

Test temperature:

Temperature range in new and old solutions monitores at the moment of renewal: 20.4-21.9 °C
The observed temperature range which was continually recorded during the test: 20.9-22.0°C

pH:

7.8-8.5 in control, 7.6-8.6 at test concentration 265.7 mg/L

Dissolved oxygen:

8.6-10.2 mg O2/L

Salinity:

Not relevant

Nominal and measured concentrations:

Nominal: 0, 5, 13.5, 36.5, 98.4 and 265.7 mg/L test substance
Measured concentrations:
Nominal 5 mg/L: mean measured concentration 4.63 mg/L (92.7% of nominal),
Nominal 265.7 mg/L: mean measured concentration 265.68 mg/L (100.0% of nominal)
All concentrations were observed to be within ± 20% of the nominal concentrations, except for one occasion, where the concentration observed of the freshly prepared sample was 70.2% of the nominal concentration. However, the concentration of the used sample of the same interval (which is the same test solution) was 86.1% of the nominal concentration. Because the measured concentration of the used sample was above 80% and the standard deviation of the control standard samples was calculated to be 0.7%, the freshly prepared sample was not re-analyzed. As all concentrations measured are close to the nominals the effect data are based on the nominal test concentrations.

Details on test conditions:

TEST SYSTEM
- Test vessel: 50 mL (nominal) glass beakers, containing approximately 50 mL of test solution and covered by glass plates
- Aeration: dilution water was aerated prior to the addition of the test substance but not during the test
- Renewal rate of test solution (frequency/flow rate): the solutions were renewed 3 times a week
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The de-ionised water used in the study contained less than 10 μg/L of copper, with a conductivity of less than 5 μS/cm and less than 2.0 mg/L NPOC-content.
- Intervals of water quality measurement:
Temperature was measured in the control solution and the highest concentration where parent daphnids were still alive, at day 0,5,10,17 and 21.
pH was measured in the control solution and the highest concentration where parent daphnids were still alive, at day 0, 3, 5, 10, 17, 19 and 21.
Dissolved oxygen concentration was measured in the control solution and the highest concentration where parent daphnids were still alive, at day 0,5,10,17,19 and 21.
Total hardness, expressed as German hardness was measured in the control solution and the highest concentration where parent daphnids were still alive, at day 0, 5, 10, 17 and 21. It was also measured as mg/L Ca2+on day 10,17 and 21.
Temperature was measured hourly in a beaker placed next to the test vessels and recorded.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16h of ambient light per day, provided by fluorescent tubes
- Light intensity:15-20 μmol/m2/s

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Animals were checked for immobilisation of parent daphnids on at least 6 days per week of the test, by gently shaking the test vessel. From the day of the first brood, observations of broods (aborted, living and dead progeny) were also made in each container at each concentration. The day of brood
release and the number of living and dead neonates per brood or abortions were noted. Any other abnormal observations were also recorded.
At the end of the test, the length of all surviving parent animals was measured to the nearest 0.1 unit using a binocular microscope (1.2 units equals 1 mm). Following measurement of body length, parent animals were placed in an oven at 105°C overnight in groups of four or five and weighed (to constant dry weight) to calculate the mean individual weight per surviving parent animals per concentration.

VEHICLE CONTROL PERFORMED: no

RANGE-FINDING STUDY
- Results used to determine the conditions for the definitive study: The test concentrations were based on an available acute study where no effect up to 100 mg/L test substance was observed.

Reference substance (positive control):

no

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

> 265.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks:

and parental length

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

>= 265.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks:

and parental length

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

>= 248.4 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Remarks:

93.5% test substance (+ impurities)

Basis for effect:

other: reproduction and parental length

Details on results:

- Mortality of parent animals:
One daphnid died in each of the following concentrations: Control (at day 19) and 13.5 mg/L (at day 17). Two daphnids died in 98.4 mg/L (at day 10 and 17). Three daphnids died in 36.5 mg/L (at day 14, 17 and 18) and 265.7 mg/L (at day 12, 14 and 21).
- No. of offspring produced per day per female:
The number of juveniles per replicate in each concentration is shown in the table included in the remarks on results. The validity criteria for the coefficient of variation (less than 25% in the control based on the number of living neonates for each parent animal alive at the end of the test) was achieved
- Body length and weight of parent animals:
Length data were found to be normally distributed and displayed homogeneity of variance. Multicomparison tests of the length of parent animals were employed. The Bonferroni t-test and the Dunett's test did not show significant effect compared to the control at any of the test concentrations, therefore the Lowest Observed Effect Concentration (LOEC) is greater than 265.7 mg/L and No Observed Effect Concentration (NOEC) is equal to or greater than 265.7 mg/L.
The average weight per Daphnia did not follow a proper dose response line but was fluctuating between the test concentrations. The test concentration of 5 mg/L would have already been significantly different from the control, but 36.5 mg/L was not. Most parent animals had eggs in their body, which was varying in number, and this could have had an influence on the fluctuation in weight.
We therefore checked (non-GLP) the influence of the presence of eggs on the weight of parent animals 21 days old and observed that animals with eggs can weigh about 35% more of the total weight of a daphnid without eggs.
The spread in weight in 21 day old animals with or without eggs is in most cases larger than the difference in weight between the test concentrations and the control and therefore weight data can not be used as a reliable endpoint. The weight data were not used for statistical evaluation of the NOEC
and LOEC because it is not possible to discriminate between effect from the test substance and eggs present in the body.
- Time to first brood release or time to hatch
The first brood releases were observed on day 10 in all test concentrations and control, but not for all 10 parent daphnids. Only in the highest test concentration all replicates released their first brood on that day, in all other concentrations some parent animals had their first brood release on day 12 of the test.
- Other biological observations:
Many of the first broods were found to be weak. Only in the highest test concentration this was not observed.
No dead young or aborted eggs were found during the test.

Results with reference substance (positive control):

Not relevant

Reported statistics and error estimates:

Data on reproduction:
The data on reproduction was tested for normality using Shapiro-Wilk's test and found to be normally distributed. The data passed Bartlett's tests for homogeneity of variance. Analysis of variance was performed on the number of living neonates per parent using the Bonferroni t-test and verified with a second multiple comparison method, the Dunnett's test.
Length data:
Multicomparison tests of the length of parent animals were employed. The Bonferroni t-test and the Dunett's test did not show significant effect compared to the control at any of the test concentrations.

Number of juveniles per parent alive at end of the test at each test concentration, mean number of neonates per concentration and coefficient of variance:

	Concentration (mg/L)
Rep no.	0	5	13.5	36.5	98.4	265.7
1	D	71	72	85	101	113
2	96	79	84	72	98	142
3	68	68	86	D	73	D
4	70	69	82	54	111	125
5	93	71	87	D	D	117
6	92	62	78	D	91	124
7	85	78	68	74	112	D
8	76	79	60	55	85	142
9	126	54	D	88	D	D
10	63	90	82	77	115	113
Mean	85	72	78	72	98	125
St. dev.	19	10	9	13	15	12
CV (%)	23	14	12	18	15	10

D = died during the test

Validity criteria fulfilled:

yes

Remarks:

<20% mortality in control group, average number of juveniles per parent in control >60 (85) in 21 days

Conclusions:

No effects up to the highest test concentration of 265.7 mg/L were found on reproduction or parental length.
The data were found to be normally distributed and homogeneous. Using Dunnett's and Bonferroni-t tests, the lowest Observed Effect Concentration (LOEC) was found to be greater than 265.7 mg/L. Based on these statistical results the NOEC is ≥ 265.7 mg/L.

Executive summary:

The purpose of this study was to assess the effect of the test substance dissolved in fresh water, on

the reproductive efficacy of Daphnia magna STRAUS - clone 5, in a semi-static test complying with the

OECD Guideline No. 211, 21 st September 1998 and EU guideline C.20 from Annex V of Directive

67/548/EEC.

The test criterion of toxicity was the reproductive capacity expressed as the number of neonates per

daphnid per day. The nominal concentrations used in the study were as follows: 0,5, 13.5,36.5,98.4 and 265.7 mg/L

As all concentrations measured are within range 80 -120% of the nominal values, the effect data are based on the nominal test

concentrations. The validity criteria were respected.

No effects were observed on reproduction and parental length. These data were found to be normally distributed and homogeneous. Using Dunnett's and Bonferroni-t tests, the lowest Observed Effect Concentration (LOEC) was found to be greater than 265.7 mg/L.

Based on these statistical results the NOEC is ≥ 265.7 mg/L. The EC50 for parent survival could not be determined.

Description of key information

There are two long term studies with aquatic invertebrates performed with L-glutamic acid, N,N-diacetic acid, tetrasodium salt (GLDA-Na4). One long term study with the freshwater phyllopoda daphnia magna and one with the marine copepoda Tisbe battagliai

 

In the fresh water study the effect of GLDA-Na4 on the reproductive efficacy of Daphnia magna STRAUS - clone 5, in a semi-static test according to OECD Guideline No. 211 was evaluated.

No effects were observed on reproduction and parental length. These data were found to be normally distributed and homogeneous. Using Dunnett's and Bonferroni-t tests, the lowest Observed Effect Concentration (LOEC) was found to be greater than 265.7 mg/L.

Based on these statistical results the NOEC is≥265.7 mg/L (249 mg/L corrected water content). The EC50 for parent survival could not be determined.

 

In the Sea water study the effect of GLDA-Na4 on the reproductive efficacy of Tisbe battagliai in a semi-static test according to ISO 14669 (1999) with modifications for a 21-day reproduction test allowing control nauplii to develop into adulthood.

For survival the median lethal concentration (EC50) was 396.2 mg/L of GLDA-Na4 (active ingredient) with 95% confidence limits between 322.3 to 486.9 mg/L over 21 days. 

The EC50 for number of nauplii was 273.7 mg/L with 95% confidence limits between 153.2 to 334.4 mg/L of GLDA-Na4 (active ingredient), the NOEC and LOEC for number of nauplii were 224 mg/L and 499 mg/L ofGLDA-Na4 respectively.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

249 mg/L

Marine water invertebrates

Marine water invertebrates

Effect concentration:

224 mg/L