L-Aspartic acid, N,N'-1,2-ethanediylbis-, sodium salt (1:3)

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method C.20 (Daphnia magna Reproduction Test)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0, 0.32, 1.0, 3.2, 10, 32 and 100 mg/L
- Sampling method: samples were taken from all test concentrations just after dosing at t = 0, 5, 7, 12, 14 and 19 days. At t = 2, 7, 9, 14, 16 and 21 days samples were taken from the same spent solutions (just before replacement). An aliquot of 10 mL was taken from each of the test vessels and pooled. Samples were then taken in 100 mL polyethylene bottles
- Sample storage conditions before analysis: formol (final concentration 1%) was used for preservation. The samples were stored in a refrigerator

Vehicle:

no

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water Flea
- Strain/clone: no data
- Justification for species other than prescribed by test guideline: no data
- Source: TNO Institute of Environmental Sciences, Schoemakerstraat 97, P.O. Box 6011, 2600 JA, Delft, The Netherlands
- Age of parental stock (mean and range, SD): One day old
- Feeding during test
- Food type: the animals were fed with cells of the unicellar alga Chlorella pyrenoidosa during the test and some yeast was added as extra food
- Amount/Frequency: the cultures were fed daily with ca. 4 x 10(9) algal cells (Chlorella) and ca. 0.13 g of yeast per 4 litre


ACCLIMATION
- Acclimation period: no data
- Acclimation conditions (same as test or not): no data
- Type and amount of food: no data
- Feeding frequency: no data
- Health during acclimation (any mortality observed): no data


METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES: once per week, all the young born in the interim period were removed

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

21 d

Post exposure observation period:

no data

Hardness:

205 mg/L expressed as CaCO3

Test temperature:

Between 19.5 and 20.6oC

pH:

Between 7.5 and 8.3

Dissolved oxygen:

The lowest value measured was 7.4 mg/L

Salinity:

no data

Nominal and measured concentrations:

Nominal concentration: 0, 0.32, 1.0, 3.2, 10, 32 and 100 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: glass, 100 mL, 50 mL of test material
- Aeration: no aeration
- Renewal rate of test solution (frequency/flow rate): no data
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): 4
- Biomass loading rate: no data


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: groundwater from a locality near Linschoten (The Netherlands)
- Total organic carbon: between 1.5 and 2.6 mg/L
- Particulate matter: no data
- Metals: Na = 1.04 mmol/L, K = 0.19 mmol/L, Ca2 = 1.35 mmol/L, Mg2 = 0.70 mmol/L
- Pesticides: no data
- Chlorine: 1.35 mmol/L
- Alkalinity: between 7.5 and 8.3
- Ca/mg ratio: 1.35/0.70
- Conductivity: between 604 and 612 microS/cm
- Salinity: no data
- Culture medium different from test medium: no data
- Intervals of water quality measurement: at the start of the test, at each replacement time, just before and just after replacement, and at the end of the test.


OTHER TEST CONDITIONS
- Adjustment of pH: between 7.5 and 8.3
- Photoperiod: 16 h light - 8 h dark regime
- Light intensity: no data


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Effects on survival and reproduction and effects on size (dry weight) or condition (swimming behaviour, colour or any other observable morphological or behavioural criterion) were visually estimated through out the 21 exposure period.

VEHICLE CONTROL PERFORMED: yes

RANGE-FINDING STUDY
- Test concentrations: four range-finding tests were carried out. The nominal tested concentrations were:
First test : 0, 32, 100, 320 and 1000 mg/L
Second test : 0, 3.2, 10, 32 and 100 mg/L
Third test : 0, 0.32 and 1.0 mg/L
Fourth test : 0, 0.032, 0.1, 0.32 and 1.0 mg/L
The range finding test was carried out with glass beakers, which contained 500 mL dilution water or test solution. Ten daphnids were placed in each test vessel at the start of the range finding test. The range finding test was carried out at a room temperature of 20 ± 1°C.
- Results used to determine the conditions for the definitive study:
The pH values in the first range-finding test varied between 7.5 and 8.2; the lowest measured oxygen concentration was 7.5 mg/L; the measured temperatures in the control were between 20.3 and 20.5°C.The pH values in the second range-finding test varied between 7.5 and 8.2; the lowest measured oxygen concentration was 8.5 mg/L
(For survival details refer to Table 5, 6 ,7 and 8.)

Reference substance (positive control):

not specified

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

ca. 32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

ca. 32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

ca. 80 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

ca. 32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: condition:colour

Details on results:

-Survival and condition - there was no mortality in the control solutions. The 21-d LC50 was estimated from the data to be greater than the highest test concentration (100 mg/L). A significant (binomial test; p = 0.05) effect with regard to survival of Daphnia magna was found at the 100 mg/L concentration. No significant mortality of Daphnia magna was found at 32 mg/L and below. Therefore, the study NOEC for mortality was considered to be 32 mg/L.

-Effects on size or condition (swimming behaviour, colour or any other observable morphological or behavioural criterion) - at 32 mg/L and below, no effects were reported, therefore this concentration can be considered the study NOEC for such effects.

-Reproduction - the 2I-d EC50 was estimated from the data to be about 80 mg/L. A relevant inhibition of reproduction was found (25% inhibition compared with the mean number of young per adult in the control) at 100 mg/L. At 32 mg/L there was no significant inhibition of reproduction (i.e. less than 25% inhibition), considered the study NOEC for such effects.

Reported statistics and error estimates:

Statistical significance for mortality was determined with a binomial test with a 95% and 99% significance level. Statistical significance for reproduction was determined with the two-tailed Dunnett-test with a 95% and 99% significance level using the mean number of young per female as observed values. In both cases the observations at each concentration were compared with those of the control.

Table 5: Number of surviving animals in the first range finding test.

Time (d)	Concentration in mg/L
	0	32	100	320	1000
0	10	10	10	10	10
14	10	8^1)	3^1)	0	0
18	10	6^1)	0	0	0

1) The test animals were smaller and paler than the control and showed no reproduction.

Table 6: Number of surviving animals in the second range finding test.

Time (d)	Concentration in mg/L
	0	3.2	10	32	100
0	10	10	10	10	10
14	10	7	4	5^1)	0
18	9	7	4	5^1)	0

1) The test animals were smaller and paler than the control and showed a decreased reproduction

Table 7: Number of surviving animals in the third range finding test.

Time (d)	Concentration in mg/L
	0	0.32	1
0	10	10	10
7^1)	9	5	7

1) All surviving test animals were in good condition.

Table 8: Number of surviving animals in the fourth range finding test.

Time (d)	Concentration in mg/L
	0	0.332	0.1	0.32	1
0	10	10	10	10	10
10^1)	10	10	10	10	10

1) All test animals were in good condition and had normal reproduction.

Table 9: Average cumulative number of young per female daphnia after 21 days exposure to various concentrations of the test substance

Nominal concentration (mg/L)	Cumulative number of young per living female
	determined	% of control
0	132	100
0.32	129	98
1	128	97
3.2	124	94
10	115	87
32	125	95
100	18*	14

Table 10: Dry weight of the parent daphnids at the end of the test

Test substance nominal concentration (mg/L)	Total dry weight (in mg)	Number of daphnids	Average dry weight per daphnia) (in mg)
0	8.8	10	0.88
0.32	7.2	9	0.8
1	6.6	10	0.66
3.2	6.5	10	0.65
10	5.3	10	0.53
32	8.6	10	0.86
100	2.6	5	0.52

Validity criteria fulfilled:

yes

Conclusions:

In a GLP study, similar to OECD Guideline 211 and EU Method C.20, the 21-d NOEC for trisodium EDDS for effects on reproduction, survival, condition and size to Daphnia magna in freshwater was 32 mg/L.

Executive summary:

In a GLP study, similar to OECD Guideline 211 and EU Method C.20, the reproductive toxicity of trisodium EDDS to the freshwater crustacean, Daphnia magna, was assessed. Ten daphnids (individually exposed) was used for each test concentration and the exposure duration was 21 d. The test was carried out under semi-static conditions with replacement of the test solutions three times per week. The nominal concentrations tested were 0, 0.32, 1.0, 3.2, 10, 32 and 100 mg/L. In addition to the quantitatively determined effects on survival and reproduction, effects on size and condition (swimming behaviour, colour or any other observable morphological or behavioural criterion) were visually estimated.

The 21-d NOEC for effects on reproduction (less than 25% inhibition), survival, condition and size was 32 mg/L, the 21-d EC50 for effects on reproduction was calculated to be 80 mg/L, and the 21-d LC50 for survival was >100 mg/L.

Description of key information

In a GLP study, similar to OECD Guideline 211 and EU Method C.20, the 21-d NOEC for trisodium EDDS for effects on reproduction, survival, condition and size to Daphnia magna in freshwater was 32 mg/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

NOEC

Effect concentration:

32 mg/L

Additional information

In a GLP study, similar to OECD Guideline 211 and EU Method C.20, the reproductive toxicity of trisodium EDDS to the freshwater crustacean, Daphnia magna, was assessed. Ten daphnids (individually exposed) were used for each test concentration and the exposure duration was 21 d. The test was carried out under semi-static conditions with replacement of the test solutions three times per week. The nominal concentrations tested were 0, 0.32, 1.0, 3.2, 10, 32 and 100 mg/L. In addition to the quantitatively determined effects on survival and reproduction, effects on size and condition (swimming behaviour, colour or any other observable morphological or behavioural criterion) were visually estimated. The 21-d NOEC for effects on reproduction (less than 25% inhibition), survival, condition and size was 32 mg/L, the 21-d EC50 for effects on reproduction was calculated to be 80 mg/L, and the 21-d LC50 for survival was >100 mg/L (Hooftman and Kauffmanvan Bommel, 1994).

No data in marine invertebrates are currently available.