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Long-term toxicity to aquatic invertebrates

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Reference
Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
April 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study performed in accordance to OECD 211 guidance
Qualifier:
according to
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Deviations:
yes
Remarks:
See method box in this RSS
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
Samples of all test concentrations were taken at during the test just before and after changing of the test solutions as well as prior to the start of the test to indicate the parent material and main degradation products concentrations. Not all samples were necessarily analysed and not all solution changes were sampled for parent material.

At least 30 mL was sampled in each case. Samples from the actual test replicates were filtered using a 0.45 µm GHP Acrodisc filter to remove algae

Parent material samples were analysed straight away and not stored so as to demonstrate the presence in the new solutions and subsequent rapid degradation of the parent material in the old solutions. This trend is predictable and repeats throughout the test. It was therefore not necessary to sample every refreshed solution for the parent material. Degradation product samples were stored in the refrigerator until analysis together with the appropriate storage stability samples.

The control and the only test concentration were analyzed at different days throughout the test. Only a limited number of analyses were conducte on the parent chemical to demonstrate the presence of the test substance at least once in each of the WAF solutions also after the longest storage period. It was known that the parent material rapidly degrades and it was for this reason that limited measurements of old test solutions took place.Analysis of the main degradation product was also not conducted on all stored samples. A selection was made to best represent the 48 and 72 hour time periods between refreshments throughout the test and calculate geometric means to best represent the average exposure.
Vehicle:
no
Details on test solutions:
The purpose of this study was to assess the toxicity 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 (OECD, 2012). Hence, the test material were left to degrade for 6 days at room temperature in the dark under slow agitation with a Teflon stirrer, and the test organisms were exposed to the partially degraded test materail.
Test organisms (species):
Daphnia magna
Details on test organisms:
The test animals were taken from a Daphnia magna clone 5 stock, (Origin: WIL Research Europe, The Netherlands) cultured in conformity with the relevant SOP. The parent animals were cultured in test medium from the day they were born.
The animals used in the test were less than 24 hours old and were obtained from parent animals reproducing parthenogenically and having an age of 2-4 weeks (having previously produced at least one brood before use). The culture is checked half-yearly for sensitivity by a reference test with potassium dichromate and is only used when guideline criteria are met.
Test type:
semi-static
Water media type:
freshwater
Total exposure duration:
21 d
Hardness:
Water hardness was 14.2 °dH which is equal to 253 mg/L as CaCO3
Test temperature:
Temperature in the test room checked with a digital thermometer ranged from: 19.6 to 21.3 °C. Temperature range observed in fresh and used solutions monitored at the moment of renewal: min. 19.7; max. 20.6 °C.
pH:
pH observed in fresh and used solutions monitored at the moment of renewal: min. 7.5; max.8.5
Dissolved oxygen:
The observed oxygen concentration range: min. 7.9; max. 10.1mg O2/l.
Nominal and measured concentrations:
The Laoding rate was 10 mg/L of the susbtance (degraded for 6 days). The analytical results demonstrated therefore that the test organisms were exposed to an average measured initial concentration of 36.0 µg/L (water solubility of the parent) and a geometric mean of 0.52 mg/L of the main degradation product.
Duration:
21 d
Dose descriptor:
NOELR
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction

 Analytical results

The concentration of the test substance present in the test vessels and the stock solutions was quantified using the methods as described in the report.

 

The aim of the study was to allow the parent material 6 x its hydrolytical half-life to degrade and assess the observed effects. This was because of the greater relevance of the degradation components to chronic aquatic toxicity. Firstly it is visible that parent material was still present in the region of its water solubility limit in test media after the degradation period. This did however not mean that no degradation had occurred. This was visible from the elevated levels of2, 4, 4-trimethylpentan-2-ol that were present in comparison to the non-degraded solution that was measured as a control due to 2, 4, 4-trimethylpentan-2-ol also being present as an impurity.

 

The analytical results demonstrated therefore that the test organisms were exposed to an average measured initial concentration of 36.0 µg/L (water solubility of the parent) and a geometric mean of 0.52 mg/L of the main degradation product.

 

The analytical measurements demonstrated behaviour of the parent material was identical to that observed in the existing acute study. That is, presence its water solubility limit at T= 0h followed by rapid degradation to <LOD at T=48h. The reason for stability of the dissolved fraction in the WAF solutions is due to the large excess of test material added (10 mg/L) when considering the extremely low solubility of the parent material (33µg/L).

 

The concentration of to 2, 4, 4-trimethylpentan-2-ol was consistently above 1 mg/L in the new (degraded) solutions and declined significantly to approximately 0.2 mg/L in the old solutions. The geometric mean of all measurements being 0.52 mg/L. It is relevant here to reference supporting study Akzo Nobel 2014 in which 2, 4, 4-trimethylpentan-2-ol caused no chronic reproductive effects to Daphnia magnaup to 2.0 mg/L, attached as a supporting study for long term toxicity to aquatic invertebrates. The analytical measurements demonstrate therefore formation of the main degradation product and hence it is fair to assume that any other water soluble degradation components would also have been present despite their measurement not being in the scope of this study.

 

Parent animal mortality

One parent animal died during the test in the control. Two parent animals died at the only test concentration. Considering that 80% survival is permitted for control performance in the test guideline and that the difference between the control and test concentration was only a single mortality this has not been considered as significant test substance related effect on the parent organisms.

 

Coefficient of variation of control fecundity

The number of juveniles per replicate in each concentration is shown in Table IV. The validity criterion 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 exceeded by 0.5 %. The mean number of neonates per parent animal alive at the end of the test and theindividual number of neonates per replicate are depicted in Figure 1 and 2, respectively..

 

 

 Statistical evaluation of the reproduction data and length and weight of parent animals at end of study

The data were found to be normally distributed using the Shapiro Wilk’s test with the exception of the dry weight data. The length and reproduction data also passed the F-Test for homogeneity of variance.

 

Reproduction

No significant difference from the control was detected using the homoscedastic T test (see figure 1 in annex 6) (See Annex 4 for raw data).

 

Length and Weight

For length data no significant difference from the control was detected using the homoscedastic T. Dry weight data was limited due to the test being conducted as a limit test. However the mean dry weight in the only test concentration was slightly higher than that of the control. Hence there was no indication of a negative influence on dry weight caused by the test substance.

 

All three endpoints indicate that there were no significant differences between the control and the only test replicate. The results can best be expressed as a NOELR (No observed effect loading rate) of 10 mg/L due to the complex nature of the degradation mixture tested.

  EC50for parent animals

An EC50based on survival of parent animals at the end of the test could not be calculated because no concentration related mortality occurred.

 

Any other biological effects observed

Weak first broods were observed for both the control and the only test concentration. This data was not statistically analysed as. The total of immobile juveniles in the control was (116) and at the test concentration (114). It was therefore concluded as a non-substance related effect.

 

Validity criteria fulfilled:
yes
Conclusions:
The following quality criteria were met:
• Cultures were in good health (i.e. disease free, no ephippia or males, no discolored animals valid reference test).
• one parent animal died in the control group over the test period, which is not more than 20%.
• The average number of juveniles per parent animal alive at the end of the test in the control was 142 after 21 days (minimum acceptable = 60)
• Analytical quality criteria were met see Table II&III

The following criterion was not met:
• The coefficient of variation in the control was exceeded by 0.5%
Executive summary:

The test substance at its water solubility limit and the accumulated degradation products resulting from a loading concentration of 10 mg/L degraded for 6 days did not have any detectable effects on the reproduction or dry weight of Daphnia magnain a 21 day chronic study under semi static conditions. It can be concluded that the water soluble degradation products as well as the parent material are not likely to display any chronic toxicity at the concentrations achievable in the environment

Description of key information

One chronic daphnia (OECD 211) was conducted using WAF approach. No effecte were observed at the water solubility limit. A supporting study (non GLP screening 21 day) has been performed on the degradation product of the regsistered susbtance, 2,4,4-Trimethyl-2 pentanol. This test showed no effects on reproduction at the highest tested concentration 2 mg/L (nominal) or 1.16 mg/L (measured mean). 

Key value for chemical safety assessment

Additional information

One GLP OECD 211 test was performed. The test substance at its water solubility limit and the accumulated degradation products resulting from a loading concentration of 10 mg/L degraded for 6 days did not have any detectable effects on the reproduction or dry weight of Daphnia magna in a 21 day chronic study under semi static conditions. It can be concluded that the water soluble degradation products as well as the parent material are not likely to display any chronic toxicity at the concentrations achievable in the environment. A supporting study (non GLP screening 21 day) was performed on one of the degradation products of the registered substance 2,4,4-Trimethyl-2 pentanol. This test showed no effects on reproduction at the highest tested concentration 2 mg/L (nominal) or 1.16 mg/L (measured mean).

In summary, the test substance is not toxic to Daphnia magna at its maximum achievable water solubility limit.