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

Long-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

fish early-life stage toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From September 11 to October 24, 1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
no data

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0, 35, 61, 109, 196, 349, 610 and 1089 mg/L
- Sampling method: samples were taken weekly from the control and the newly prepared media from all the test substance concentrations, and after 48 h from the same spent media at replacement of the media.
- Sample storage conditions before analysis: formaldehyde was added (final concentration 1%) for preservation of the samples.

Vehicle:

no

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebra fish
- Strain: no data
- Source: commercial tropical fish hatchery M.B. Ruijsbroek B.V. (Noordvliet 159, Maassluis), The Netherlands
- Pre-exposure reproductive performance: no data


METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
- Numbers of parental fish (i.e. of females used to provide required number of eggs): no data
- Method of collection of fertilised eggs: As soon as the eggs were deposited they were collected manually and put into the test vessel
- Subsequent handling of eggs: the dead eggs or larvae were counted and removed at each replacement time; the survivors were also counted and their size and condition (swimming behaviour, presence of malformations, or any other visually observable morphological or behavioural criterion) was compared with that of the control animals.

POST-HATCH FEEDING
- Start date: from Day 10
- Type/source of feed: Artemi nauplii enriched with Selco as main food addition to rotifers (mass laboratory cultures)
- Amount given: no data
- Frequency of feeding: no data

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

30 d

Post exposure observation period:

no data

Hardness:

210 mg/L of CaCO3

Test temperature:

24.7 to 25.6oC (refer to Table 2 for details)

pH:

Between 7.1 to 8.1

Dissolved oxygen:

The lowest measured value was 7.2 mg/L

Salinity:

no data

Nominal and measured concentrations:

0, 32, 56, 100, 180, 320, 560 and 1000 mg/L (nominal), equates to 0, 35, 61, 109, 196, 349, 610 and 1089 mg/L when corrected for the actual purity of the test substance.

Details on test conditions:

TEST SYSTEM:
- Test vessel:
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: glass
- Aeration: slightly aerated
- No. of fertilized eggs/embryos per vessel: 40
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Biomass loading rate: no data


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: ground water from locality near Linschoten (The Netherlands)
- Total organic carbon: refer to Table 1
- Particulate matter: refer to Table 1
- Metals: (trace metals): refer to Table 1
- Pesticides: no data
- Chlorine: refer to Table 1
- Alkalinity: 8-8.2
- Ca/mg ratio: refer to Table 1
- Conductivity: no data
- Salinity: no data
- Culture medium different from test medium: no data
- Intervals of water quality measurement:begining and at the end of the test, also at each replacement time in fresh media and weekly in the spent media.


OTHER TEST CONDITIONS
- Adjustment of pH: betwen 7.1 and 8.1
- Photoperiod: 16 h light - 8 h dark
- Light intensity: no data


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :


VEHICLE CONTROL PERFORMED: no


RANGE-FINDING STUDY
- Test concentrations: no
- Results used to determine the conditions for the definitive study: no data

Reference substance (positive control):

not specified

Duration:

21 d

Dose descriptor:

LC50

Effect conc.:

> 1 089 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

25 d

Dose descriptor:

LC50

Effect conc.:

ca. 1 008 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: >743-<1372

Duration:

27 d

Dose descriptor:

LC50

Effect conc.:

ca. 892 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: >671-<1187

Duration:

30 d

Dose descriptor:

LC50

Effect conc.:

ca. 791 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: >607-<1031

Duration:

30 d

Dose descriptor:

NOEC

Effect conc.:

ca. 61 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

30 d

Dose descriptor:

NOEC

Effect conc.:

ca. 61 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

morphology

Duration:

30 d

Dose descriptor:

NOEC

Effect conc.:

ca. 348 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

length

Duration:

30 d

Dose descriptor:

NOEC

Effect conc.:

ca. 196 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

weight

Details on results:

- Hatching and mortality (see Table 4 for details):
At day 6, all eggs (except one egg at 348 mg/L and one dead egg at 1089 mg/L) were ultimately hatched into healthy larvae without visible malformations, however, at the concentrations above 196 mg/L some eggs hatched with a small delay during this 6 d period.
At the end of the test (day 30), one out of eighty test animals was dead in the control and 32 mg/L group, and 2 dead at 61 mg/L. A significant effect (p = 0.05; binomial test) with respect to survival at day 30 was seen in the 109 mg/L (24% mortality) and above groups (except at 348 mg/L with only 6% mortality at day 30). The NOEC and LOEC for mortality were therefore 61 and 109 mg/L, respectively.

- Visual observations:
In the control media and at the concentrations of up to 61 mg/L the surviving larvae swam and fed actively during the exposure period and no malformations were noted during the test (except a few fish "being small" at the lower test concentrations and in the controls; they were considered to be incidental cases and were not taken into account for the estimation of the NOEC and LOEC). The NOEC and LOEC values for condition were therefore 61 and 109 mg/L, respectively.

- Growth (see Table 4 for details):
At 610 and 1089 mg/L, the lengths of the test fish were significantly (p = 0.01) lower at 1.04 and 0.77 cm, respectively, compared with the controls (length of 1.2 cm). The highest concentration tested without a significant effect on growth (measured as dry weight) was 196 mg/L. At 348, 610 and 1089 mg/L, growth (mean average dry weight per fish) was significantly retarded (1.59, 1.55 and 1.87 mg, respectively) for the surviving fish compared with controls (2.45 mg). Therefore, the NOEC and LOEC with respect to growth were 196 and 348 mg/L, respectively.

Results with reference substance (positive control):

No data

Reported statistics and error estimates:

Statistical significance for mortality was determined with a binomial test at the 95% significance level, combining the results of the quadruplicates. Statistical significance for growth was determined with the two-tailed Dunnett-test with a 95 and 99% significance level. In both cases the observations at each concentration were compared with those of the control. In case of significance at the 99% level only that level is given.

Table 3: Results of the early life stage test with trisodium EDDS

Parameter (d)	Effect	Nominal concentration (mg/L) (95% confidence interval)
		Without correction for trisodium EDDS^1)	After correction for trisodium EDDS%^2)
6-d LC50	mortality	>1000	>1089
21-d LC50	mortality	>1000	>1089
25-d LC50	mortality	926 (682-1260)	1008 (743-1372)
27-d LC50	mortality	819 (616-1090)	892 (671-1187)
30-d LC50	mortality	726 (557-947)	791 (607-1031)
30-d NOEC	mortality	56	61
30-d LOEC	mortality	100	109
30-d NOEC	condition	56	61
30-d LOEC	condition	100	109
30-d NOEC	growth (length)	320	348
30-d LOEC	growth (length)	560	610
30-d NOEC	growth (dry weight)	180	196
30-d LOEC	growth (dry weight)	320	348

 1) 38.6%

2) 42.04%

 

Table 4: Summary of results on hatching, mortality and growth of eggs/larvae of Brachydanio rerio exposed to several concentrations of trisodium EDDS

Nominal concentration of trisodium EDDS (mg/L)^1)	% of eggs hatched after 6 d	% mortality after 30 d	No. of fish	Length^2)(cm)	Mean of the average dry weight per fish^2)(mg)
0	100	1	79	1.20 ± 0.15	2.45 ± 0.29
32	100	1	79	1.17 ± 0.19	1.89 ± 0.22
56	100	3	78	1.19 ± 0.17	1.79 ± 0.43
100	100	24^3)	61	1.25 ± 0.16	2.31 ± 0.48
180	100	25^3)	60	1.22 ± 0.21	2.02 ± 0.40
320	99	6	75	1.14 ± 0.15	1.59 ± 0.13^5)
560	100	36^3)	51	1.04 ± 0.16^4)	1.55 ± 0.55^5)
1000	96	83^3)	14	0.77 ± 0.18^6)4)	1.87 ± 1.00^6)

 1) Concentration without correction for the actual measured trisodium EDDS.

2) Mean and standard deviation (dry weight calculated as explained in the text).

3) Mortality significantly higher than that of the control animals (binomial test; p = 0.05).

4) Significantly less than control (two-tailed Dunnett-test. p = 0.01).

5) Significantly less than control (two-tailed Dunnett test. p = 0.05).

6) Only 14 fish survived at this concentration; the dry weight of the fish from only one replicate could be determined accurately and is given in this table.

 

Validity criteria fulfilled:

yes

Conclusions:

In a GLP study conducted according to OECD Guideline 210, the effect on hatching, survival and the occurrence of egg and larval malformations following exposure to trisodium EDDS in the freshwater Zebra fish, Brachydanio rerio, was investigated. At nominal concentrations of 348 mg/L and above, growth (measured as dry weight) was significantly retarded for the surviving fish, but no effects on growth were seen at 196 mg/L (considered the 30-d NOEC for such effects) and below. The 30-d NOEC for mortality and condition was 61 mg/L.

Executive summary:

In a GLP study conducted according to OECD Guideline 210, the influence of trisodium EDDS on hatching, survival and the occurrence of egg and larval malformations in the fresh water fish, Brachydanio rerio. The test was started with a number of 4 x 20 eggs at each test concentration and the exposure duration was 30 d. The test was carried out as a semi-static test, with replacement of the test solutions and control media every 48 h. The nominal concentrations of trisodium EDDS tested were 0, 35, 61, 109, 196, 348, 610 and 1089 mg/L. During the test, samples were taken from the control and the newly prepared test media and after 48 h from the same spent media and from the stock solutions.

At day 6, all eggs (except one egg at a nominal test substance concentration of 348 mg/L and one dead egg at 1089 mg/L) were ultimately hatched into healthy larvae without visible malformations, however, at the measured concentrations above 196 mg/L some eggs hatched with a small delay during this 6-d period. At the end of the test, one out of eighty test animals was dead in the control and there was a significant effect with respect to survival at 109 mg/L and above (except at 348 mg/L). At a measured concentration of 61 mg/L, there was no significant effect on survival. In the control media and at the concentrations of up to 61 mg/L the surviving larvae swam and fed actively during the exposure period and no malformations were noted during the test, except a few fish in the control and in the lower concentrations were small (these were considered to be incidental cases and were not taken into account for the estimation of the NOEC and LOEC).

At 348 mg/L and above, growth (measured as dry weight) was significantly retarded for the surviving Zebra fish, but no effects on growth were seen at 196 mg/L (considered the 30-d NOEC for such effects) and below. The 30-d NOEC for mortality and condition was 61 mg/L.

Description of key information

In a GLP study conducted according to OECD Guideline 210, the effect on hatching, survival and the occurrence of egg and larval malformations following exposure to trisodium EDDS in the freshwater Zebra fish, Brachydanio rerio, was investigated. At nominal concentrations of 348 mg/L and above, growth (measured as dry weight) was significantly retarded for the surviving fish, but no effects on growth were seen at 196 mg/L (considered the 30-d NOEC for such effects) and below.

The 30-d NOEC for mortality and condition was 61 mg/L.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

NOEC

Effect concentration:

61 mg/L

Additional information

In a GLP study conducted according to OECD Guideline 210, the influence of trisodium EDDS on hatching, survival and the occurrence of egg and larval malformations in the fresh water fish, Brachydanio rerio. The test was started with a number of 4 x 20 eggs at each test concentration and the exposure duration was 30 d. The test was carried out as a semi-static test, with replacement of the test solutions and control media every 48 h. The nominal concentrations of trisodium EDDS tested were 0, 35, 61, 109, 196, 348, 610 and 1089 mg/L. During the test, samples were taken from the control and the newly prepared test media and after 48 h from the same spent media and from the stock solutions.

At day 6, all eggs (except one egg at a nominal test substance concentration of 348 mg/L and one dead egg at 1089 mg/L) were ultimately hatched into healthy larvae without visible malformations, however, at the measured concentrations above 196 mg/L some eggs hatched with a small delay during this 6-d period. At the end of the test, one out of eighty test animals was dead in the control and there was a significant effect (p = 0.05) with respect to survival at 109 mg/L and above (except at 348 mg/L). At measured concentrations of 61 mg/L, there was no significant effect on survival. In the control media and at the concentrations of up to 61 mg/L the surviving larvae swam and fed actively during the exposure period and no malformations were noted during the test, except a few fish in the control and in the lower concentrations were small (these were considered to be incidental cases and were not taken into account for the estimation of the NOEC and LOEC).

At 348 mg/L and above, growth (measured as dry weight) was significantly retarded for the surviving fish, but no effects on growth were seen at 196 mg/L (considered the 30-d NOEC for such affects) and below. The 30-d NOEC for mortality and condition was 61 mg/L (Hooftman and van Drongelen-Sevenhuijsen, 1997).

No data in marine fish are currently available.