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Long-term toxicity to fish

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fish early-life stage toxicity
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study conducted in compliance with GLP regulations
according to guideline
OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)
Principles of method if other than guideline:
The study was performed according to the OECD Guideline for Testing of Chemicals No. 210, adopted July 17, 1992 ("Fish Early-Life Stage Toxicity Test") and the (U.S.) EPA-FIFRA 72-4 (a), 1982 requirements considering the SEP (Standard Evaluation Procedure) "Fish Early Life-Stage Test", by M. Rexrode and T.M . Armitage, U.S. EPA, Office of Pesticide Programs, Hazard Evaluation Division, EPA 540/9-86-138, July 1986.
GLP compliance:
Analytical monitoring:
Details on sampling:
- The stability of the test substance in water was verified by weekly concentration control analysis.
- Analytical verification: Samples were taken on day 0 and on day 7 before renewal of the test solutions from all test vessels.
- Before the transfer to the flow through system on day 11 samples were taken representatively only from 1 replicate per test group.
- During exposure in the flow through system samples were taken at weekly intervals alternating from one of the 4 replicates of each group, generally before the replacement of the stock solutions, and were analyzed for content of test substance.
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
Test species: Zebra fish
Strain: Brachydanio rerio HAM. and BUCH.
Supplier: Zierfischcenter Kloeckner, Ludwigshafen, Germany

- Type/source of feed: Beginning at the end of hatch the larvae were offered newly hatched brine shrimp larvae (artemia naupli) and a special diet for newly hatched fish ("AZ 100" supplied by Tetramin, Tetrawerke, Melle, Germany).
- From study day 27 on "Tetraminbaby", another special commercial diet for newly hatched fish from the same supplier was fed additionally.
- From study day 30 on "Tetraminbaby" was replaced by the normal commercial fish diet "Tetramin".
- Feeding with ,AZ 100" was stopped on study day 31 and only "Tetramin" was fed during the rest of the study.
- Frequency of feeding: Feeding was done at least twice daily on workdays and once daily on weekends and was increased in quantity with the duration of the study and thus with the size of the fish.
- Feeding was continued until one day before the termination of the study.
Test type:
Water media type:
Limit test:
Total exposure duration:
35 d
Nominal and measured concentrations:
The OECD and EPA Guidelines request the testing of at least 5 concentrations spaced by a "constant factor" of 3.2 (OECD) or by the constant factor 0.5 (EPA) and a control without test substance. One of the selected concentrations should impair a life-stage, and another test concentration should be without any adverse effect on the life-stages (NOAEL = no observed adverse effect level).
The following concentrations were selected for this study on the basis of available acute aquatic toxicity data: 0.0, 1.1, 3.3, 7.7, 16.4 and 35.1 mg/L. The corresponding concentrations of the EDTA-anion were 0.0, 0.8, 2.3, 5.4, 11.5 and 24.6 mg/L.
The mean analytically determined concentration values of the test substance in the test water were generally within a range of +/- 20 % of the nominal concentrations.
The mean measured concentrations were (% of nominal):
1.0 mg/L = 90.9 %,
3.2 mg/L = 97.0 %;
7.8 mg/L = 101.3 %;
16.8 mg/L = 102.4 %
36.9 mg/L = 105.1 %
of the nominal values.
Details on test conditions:
- Aeration: no aeration
- Photoperiod: The aquaria were lighted by fluorescent tubes with a mean light intensity at the water surface of 540 Lux during the semistatic part and 164 Lux during the flow-through part.
- The photoperiod was automatically maintained and was 16 hours light and 8 hours darkness.
- Test vessels: open aquaria
35 d
Dose descriptor:
Effect conc.:
>= 25.7 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Details on results:
In conclusion, under the conditions of this study, the overall NOAEC (no observed adverse effect concentration) was >= 35.1 mg/L (nominal concentration) and >= 36.9 mg/L (based on the mean analytically determined concentration) and the lowest concentration with adverse effects (LOAEC) was > 35.1 mg/L (nominal concentration) and > 36.9 mg/L (based on the mean analytically determined concentration).

Based on the content of the EDTA-anion and on the mean of the analytically determined concentrations the NOAEC was >= 25.7 mg EDTA/L.
The survival at start of hatch was slightly lower in all concentration groups (84 - 87 % vs. 91% in the control).
With only one of the 3 statistical methods used the deviations in the 2 highest concentration groups were statistically significant . There was, however, no concentration effect relationship and the differences to the control group were < 10 % in all groups and the survival in all test groups was within a range which would be generally regarded as acceptable for control groups. The deviations are therefore considered to be accidental and not caused by the test substance.
Compared to the control group survival from start of hatch to the termination of hatch (day 1 - 5) was statistically significantly reduced in the
concentration group 3 (7.7 mg/L) using the Wilcoxonon-test and additionally in concentration group 2 (3.3 mg/L) using the log-rank test, but there was no statistically significant deviation using Fisher's exact test and also no deviation from the control group in the 2 highest concentration groups (16.4 and 35.1 mg/L). No concentration dependent tendency was observed. The deviations were not considered to be caused by the test substance.
Survival from the end of the hatch to the end of the study (day 5 - 35) was not statistically significantly decreased in comparison to the control group in any of the concentration groups 2 - 5 (3.3 - 35.1 mg/L).
Since one-sided statistical methods are used, increases can not be statistically significant.

Over the whole study period (day 0 - 35) no statistically significant decreases in survival were observed in any of the concentration groups in comparison to the control
group. The overall survival rates were 65 % in the control group and 57, 63, 70, 63 and 70 % in the concentration groups 1 - 5, respectively.
The survival rate in the highest dose group was 7.7 % higher than in the control group. No concentration related tendency can be seen. This supports the conclusion that the statistically significant differences to the control group observed in the first 5 days are not substance-related, but are caused by normal variations.

In conclusion, the NOAEC for survival is >= 35.1 mg/L (nominal concentration) and >= 36.9 mg/L (based on the mean analytically determined
concentrations), the LOAEC is > 35.1 mg/L (nominal concentration) and > 36.9 mg/L (based on the mean analytically determined concentrations).

Hatch in the replicates of the control group started at days 1 - 3 and was completed on days 4 - 5.
In all concentration groups hatch started nearly simultaneously with the control group on day 1 and was completed on days 3 - 5.

The first swim-up was observed in one replicate of group 5 on day 4 and in all othern replicates of the concentration and control groups on day 5.
Swim-up in all test groups was completed on day 5.

In conclusion a no substance related effect was observed on the time to hatch and swim-up in any of the tested concentration groups up to 35.1 mg/L.

In the control group as well as in the concentration groups occasionally deformations, reduced growth and swimming in circles was observed in individual animals. In groups
2 and 4 additionally swimming at the water surface and lying on the bottom of the test vessel was observed in single animals, respectively. No relevant differences were
observed between the concentration groups and the control group.

Thus, the NOAEC for sublethal effects is >= 35.1 mg/L (nominal concentration) and >= 36.9 mg/L (based on the mean analytically determined concentration), the LOAEC is
> 35.1 mg/L (nominal concentration) and > 36.9 mg/L (based on the mean analytically determined concentration).

No effects on body weight and length were seen in any of the concentration groups.

Thus, the NOAEC for the impairment of body weight and length was >= 35.1 mg/L (nominal concentration) and >= 36.9 mg/L (based on the mean analytically determined concentration).
The LOAEC for the impairment of growth was > 35.1 mg1L (nominal concentration) and > 36.9 mg/L (based on the mean analytically determined concentration).

Description of key information

No chronic data are available for EDTA-Zn(NH4)2. One reliable 35 d early life stage study on chronic toxicity to zebrafish (danio rerio) of the substance EDTA-CaNa2 is available from BASF. This is therefore the key study. The NOEC is >= 35.1 mg/L based on nominal concentration and recalculated as EDTA-Zn(NH4)2 the NOEC >= 36.6 mg/L .

Key value for chemical safety assessment

Fresh water fish

Fresh water fish
Dose descriptor:
Effect concentration:
36.6 mg/L

Additional information

Since no chronic adverse effects have been determined in this study the NO(A)EC is a >= value, which can't be expressed in the box above. For EDTA- Zn(NH4)2, the recalculated NOAEC is >= 38.4 mg/L, based on mean measured concentration, but >= 36.6 mg/L based on nominal concentration.