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Long-term toxicity to fish

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fish early-life stage toxicity
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to guideline
OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)
Version / remarks:
adopted in 1992
GLP compliance:
Specific details on test material used for the study:
- Batch No.: Lot OF1 201 D6C4
- Supplier: DOW Deutschland Inc.
- Physical state / appeararce: solid / white
- Content of active ingredient: main component 90.5 % (w/w), water 9.6 % (w/w) (Analytical Report ZAX 00L00386)
- Homogeneity: Homogeneous (Analytical Report ZAX 00L00386)

- Storage stability: The storage stability of the test substance over the study period has been verified by analytical recharacterization (Analytical report ZAX 01LOOO15).
- Storage conditions: At room temperature, with exclusion of air
- Solubility in water: Approximately 100 g/L
- The analytical examinations of the test substance were conducted at the Analytical Department, BASF AG.
Analytical monitoring:
Details on sampling:
- The stability of the test substance in water was verified by weekly concentration control analyses.
- 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.
Details on test solutions:
- Method: The test solutions for the semistatic part were prepared by dilution of a stock solution with dilution water. The stock solution was prepared by weighing 0.5 g test substance into a volumetric flask which was than made up with dilution water to a volume of 1 L and thoroughly mixed. The stock solution was diluted with dilution water in a 5 L volumetric flask. The solution was poured into the test vessels. Stock solutions for the flow through part were prepared separately for each concentration group. For preparation of the stock solutions the test substance was weighed, transferred into a 20L-aquarium and made up to a volume of 20 L with dilution water. The mixture was stirred with an ultra turrax stirrer and transferred into the stock solution tanks. The test solutions in the semistatic part and the stock solutions in the flow through part were prepared and replaced once weekly. The preparation of the first stock solution was done on study day 0.

Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
- Common name: zebra fish
- Source: Zierfischcenter Kloeckner, Ludwigshafen, Germany

- Numbers of parental fish (i.e. of females used to provide required number of eggs):
- Method of collection of fertilised eggs: In the morning glass trays covered with wire grating with green glass trees on the top were placed into the aquaria to induce spawning and for collection of eggs. These spawning trays were removed after some hours and the number of eggs was estimated.
- Subsequent handling of eggs: The intact clear coloured freshly fertilized eggs were removed from the tray, washed several times with dilution water in petridishes. Eggs from both trays were put together in one group from which eggs were taken and
transferred randomized to beakers with the test solutions. They were placed into the exposure chambers within 2 hours after the spawning tray had been removed and within about 4 hours after fertilization. The embryos of 5 representative eggs per tray were examined for their developmental stage. All embryos were in a stage below the 32 cell stage, most of them in the 8 cell stage.

- 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.
- Feeding frequency: 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
90 - 100 mg/L as CaCO3
Test temperature:
25 +- 0.5 °C
7.9 - 8.2
Dissolved oxygen:
6.4 - 8.0 mg/L
242 - 270 µS
Nominal and measured concentrations:
Nominal: 0.0, 1.1, 3.3, 7.7, 16.4 and 35.1 mg/L. The corrseponding concentrations of the EDTA anion were 0.0, 0.8, 2.3, 5.4, 11.5 and 24.6 mg/L
Measured: over 90% of nominal
Details on test conditions:
- Test vessel: glass aquaria (40 cm x 10.5 cm x 30 cm) with an overflow, which was covered with a plastic gauze containing a water volume of 8 L
- Type (delete if not applicable): covered with transparent lids
- Aeration: no
- Type of flow-through (e.g. peristaltic or proportional diluter): The flow rates of the test solutions or the water of the control during the flow through part were 10 L/hour/concentration or control group . The water flow from the mixing tank was split by an "udder' into 4 equal parts for the 4 replicates of each test group . Thus, each test aquarium was supplied by 2 .5 liters test water/hour . Considering the size of the aquaria, the theoretical exchange rate of the water contents was approximately 7.5 exchanges per 24 hours .
- Renewal rate of test solution (frequency/flow rate): daily (semistatic part)
- No. of fertilized eggs/embryos per vessel: 25
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

- Source/preparation of dilution water: mixture of tap water and deionized water of about 3:5. The tap water was non-chlorina~,ed drinking water obtained from the municipal water works of the city of Frankenthal, purified through a charcoal filter
- Culture medium different from test medium: no
- Intervals of water quality measurement: Temperature was measured daily. DO and pH were measured twice weekly. Hardness during the semistatic part was conducted with dilution water in an additional beaker and during flowthrough part with dilution water from the water supply.

- Adjustment of pH: no
- Photoperiod: 16 hours light and 8 hours darkness
- Light intensity: 540 Lux during the semistatic part and 164 Lux during the flow-through part.

- embryo survival day 0 - 1 (until beginning of hatch )
- embryo/sac fry survival days 1 - 5 (beginning to end of hatch )
- survival of young fish days 5 - 35 (end of hatch until end of study)
- survival of individuals from days 0 - 35 (beginning to termination of the study) 0
Mortality was determined daily. Dead test organisms were removed when first observed. Dead embryos could be identified by the white colour of the eggs. Dead sac-fry larvae or fish were identified by their immobility when being touched slightly with a rod at the basis of the caudal peduncle and no movement of the operculae (no breathing) could be observed.
Time to hatch and swim-up
- The time spans from the initiation of the study to the onset of hatch and until termination of hatch were determined.
- The time span from termination of hatch to termination of swim-up was defined . 0
Toxic signs (symptoms) and abnormalities
The occurrence of sublethal effects, if any, in sac-fry larvae and young fish, such as loss of equilibrium, erratic swimming, loss of reflex, exitability, discolouration or change of behaviour, etc. were determined visually generally at least on workdays and recorded. Any observed abnormalities were recorded .
Body weight and length
The individual body weights (wet weight) and individual body lengths (total length, from the tip of the snout to the distal end of the caudal fin) were determined at the end of the study. About 1 day before being weighed the feed was withdrawn . The body length was determined with vernier calipers.

- Begin of post-hatch period: 13 days after hatch had been completed in all test groups the test organisms were transferred from the beakers with sernistatic exposure to the aquaria of the flow through system. The number of test organisms was not reduced.
Reference substance (positive control):
Key result
35 d
Dose descriptor:
Effect conc.:
>= 35.1 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
other: all considered endpoints
Remarks on result:
other: nominal concentration analytically verified
Details on results:
In conclusion, under the conditions of this study, the overall NOEC 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 NOEC 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. However, no concentration effect relationship was observed 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 cannot 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 other 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 were 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).

Validity criteria fulfilled:
The substance is not harmful to fish at chronic exposure.
fish early-life stage toxicity
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Category justification document is attached in chapter 13.
Reason / purpose for cross-reference:
read-across source
Key result
35 d
Dose descriptor:
Effect conc.:
>= 25.7 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
other: all endpoints considered

Description of key information

NOEC (21d) >= 35.1 mg/L for all relevant endpoints of Danio rerio (OECD 210, read across)

Key value for chemical safety assessment

Additional information

Since no studies are available investigating the long-term toxicity of Tetrasodium ethylenediaminetetraacetate (CAS No. 64-02-8) to fish, the assessment was based on a study conducted with subcategory 1a member Sodium calcium edetate (CAS No. 62-33-9) in a read across approach. This is in accordance with Regulation (EC) No. 1907/2006, Annex XI, 1.5. Grouping of substance and read across approach. Further justification for suitability of read across between Subcategory 1 members is given within the category justification attached to chapter 13.

The key study was performed according to OECD guideline 210 in compliance with GLP using Danio rerio as test organism (BASF 2001). Concentrations between 1.1 and 35.1 mg/L were tested in a flow through design. The stability of the test substance throughout the test was verified by weekly concentration control analyses. The survival at start of hatch was slightly lower in the treatments but not significantly different to the control (84 -87% vs 91% in the control). No substance related effect was observed on the time to hatch and swim-up in any of the tested treatments up to 35.1 mg/L. Thus, the 35d-NOEC is determined to be >= 35.1 mg/L based on nominal concentrations. Based on the suitability of the read across approach this conclusion can also considered to be true for Tetrasodium ethylenediaminetetraacetate (CAS No. 64-02-8).