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Long-term toxicity to aquatic invertebrates

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Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
07-2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: study is done under GLP and considered complete
Qualifier:
according to
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Vehicle:
no
Details on test solutions:
All test solutions were prepared separately. Specific loading rates of the test substance were added to M7 medium and stirred for 48 hours. The resulting mixtures were stabilized for 24 hours in a separation funnel after which the Water Accommodated Fractions (WAFs) were
obtained using the drain cock.
Test organisms (species):
Daphnia magna
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Details on test conditions:
The reproduction test was performed in closed vessels in a semi-static system. It included 10 vessels per test concentration and 20 vessels for an untreated control group. Each of the vessels contained one neonate «24h old) Daphnia magna in 50 ml test medium. The nominal
concentrations of Trigonox 301 tested were 1.0, 1.8, 3.2, 5.6 and 10 mg/L. The study duration was 21 days and the test solutions were renewed every 48 hours. The daphnids were fed on a daily basis with a Chtorella pyrenoidosa suspension.
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
3.1 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
> 2.7 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
reproduction
Details on results:
Two out of the twenty parental daphnids in the control died during the test period. Mortality in the Trigonox 301 treatments of 0.93, 1.8 and 2.7 mg/l was comparable to or lower than the control and not statistically significant. 90-100% of the daphnids exposed to 4.9 and 8.3 mg/L, respectively, died during the test. Hence, the mortality of the parental daphnids was clearly treatment related and followed a very steep dose curve.

The average cumulative number of young per female in the control after 21 days was 120 ± 13. The reproduction curve recorded at 0.93, 1.8 and 2.7 mgtl closely followed or was even slightly above the curve of the control. The curves recorded at the higher concentrations clearly stayed behind from the beginning of reproduction. Since these higher concentrations of 4.9 and 8.3 mg/L showed a high mortality number of parental organisms no final judgement regarding reproduction was possible. There were no recordings of unhatched (aborted) eggs at any of the test concentrations throughout the 21-day test period. Immobile young were only recorded at 4.9 mg/L.

Validity criteria fulfilled:
yes
Conclusions:
study is complete and valid
Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Deviations:
yes
Remarks:
Additional analysis and extraction methods were used to better demonstrate exposure at the 4.88 mg/L under identical conditions to the test.
Principles of method if other than guideline:
At the start of the test the daphnids were randomly distributed to the test vessels. For each test concentration and control, ten vessels, with one daphnid per vessel were tested. Parent animals were checked for immobilisation on at least 5 days per week of the test, by gently shaking the test vessel. From the day of the first brood, observations of broods (aborted, living and dead progeny) were also made in each container at each concentration. The day of brood release and the number of living and dead neonates per brood or abortions were noted. Any other observations of abnormalities were also recorded.

At the end of the test, the length of all surviving parent animals was measured to the nearest 0.1 unit using a binocular microscope (one unit was equal to = 1.6 mm). Following measurement of body length (including spine), parent animals were placed in an oven set at 105 °C overnight in groups and then weighed to allow calculation of the mean individual weight per surviving parent animal per concentration. The test duration was 21 days.

After the main test an additional study was conducted using the 50 mg/L test concentration only. Procedures were identical to that as described in the normal test but with additional analytical measurements on the filtered algae component to obtain a better representation of the exposure concentrations. Replicates of the test medium with and without algae and with algae and test organisms were investigated for a 72h period. A control without test material with and without algae was also made as an analytical blank.
After the determination of the 21d NOEC and when considering the additional data from the 1st additional study further analysis at the reproduction NOEC of 4.88 mg/L was conducted in a second additional study. This involved repeating testing using identical procedures at 4.88 mg/L using 2 week old daphnia for a one week period. The new, old (48h) and old (72h) samples were subjected to exhaustive extraction and analytical methods to better quantify the test substance and the exposure that actually occurred at the NOEC. All analysis was conducted in duplicate.
GLP compliance:
no
Analytical monitoring:
yes
Details on sampling:
Samples of all new and old test concentrations and stock solutions were taken during the test. A limited number of these were selected for chemical analysis based on the biological results. For the grazing study samples of the test material at 15.3 mg/L were taken for all vessels at 0, 24, 48 and 72 hours. Control was only taken at the T=0 to generate appropriate background response.
Vehicle:
no
Details on test solutions:
Preparation of the stock solutions
Due to the test material being a mixture of relatively low solubility and a mixture was prepared as a WAF. Stock solutions in the normal manner were therefore not made.

Preparation of the test solutions
Each test concentration was weighed separately and added to up to 2 liters of test medium. The resulting solutions were stirred as slowly as the stirring apparatus allowed for at least 24 hours. After which the solutions were left for 1 hour to stabilize possible emulsions after which the water accommodated fraction was ‘’ tapped’’ from the WAF vessel and used directly. The WAF vessels were designed as such that floating or sunken test material is not transferred and only the water accommodated fraction reaches is removed and tested. Stirring was in some cases up to 72 hours. Analytical measurements demonstrate that this was not of negative consequence to the test material concentrations.
Test organisms (species):
Daphnia magna
Details on test organisms:
The test animals were taken from a Daphnia magna clone 5 stock, (Origin: Charles River Laboratories, 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 an appropriate reference toxicant. The most recent reference test results were within the range given in the test guideline. Furthermore there was no abnormal mortality or winter egg formation. The culture was therefore deemed suitable for use.

Test animals were fed a diet of 0.1 to 0.2 mg carbon per daphnid per week day (in 50 mL) or 0.2 to 0.4 mg (in 100 mL) (Friday to Sunday) during weekends, in the form of the algal strain Chlorella vulgaris. The strain is cultured in the Environmental Chemistry laboratory and total organic carbon content has previously been measured.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Test temperature:
20.8-21.6 °C
pH:
7.5-8.3
Dissolved oxygen:
7.7-9.1 mg/L O2
Nominal and measured concentrations:
nominal test concentrations: 0.48, 1.53, 4.88, 15.3 and 50 mg/L including a test medium only control.
Details on test conditions:
TEST SYSTEM
- Test vessel: 50 mL (nominal) glass beakers were used during weekdays, containing approximately 50 mL of test solution and covered by glass plates during the test. For the weekend 100 mL beakers were used containing approximately 100 ml of test medium so as to ensure sufficient food for the weekend period.
- Type (delete if not applicable): open
- Aeration: No
- Renewal rate of test solution (frequency/flow rate): 3 times per week
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10

TEST MEDIUM / WATER PARAMETERS
Test medium: M4 medium (Elendt 1990)
- Culture medium different from test medium: No

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16h light
- Light intensity: ambient light per day, provided by “day light” fluorescent tubes.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Reproduction, parental length, weight and mortality

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
1.6 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
reproduction
Details on results:
Parent animal mortality
One parent animal died in the control and at 4.88, 15.63 and 50.0 mg/L, Three died at 0.48 mg/L and, two died at 1.53 mg/L. The mortality was not concentration related and was therefore not factored into the reproduction figures.

Reproduction (Primary endpoint)
A NOEC and LOEC for reproduction for the substance as nominal test material were determined as 4.9 and 15.6 mg/L respectively. The EC10 for reproduction was calculated as 4.1 mg/L. The NOEC for reproduction can also be expressed as 1.6 mg/L (Time weighted measured mean)

Length
A NOEC and LOEC for length for the substance as nominal test material were determined as 1.5 and 4.9 mg/L respectively. The EC10 for length was calculated as 28.3 mg/L.

Weight
Sufficient replicates for a valid statistical analysis could not be generated by this data. When reviewing the data manually without the use of statistical software overlap to the control values is seen up until 15.6 mg/L after which weight in the highest concentration is clearly reduced. The NOEC is therefore estimated as 15.6 and LOEC as 50.0 mg/L

EC50 for parent animals
An EC50 based on survival of parent animals could not be determined as no significant adult mortality occurred in the highest test concentration. The EC50 for adult mortality can therefore only be expressed as > 50 mg/L based on Nominal concentrations.
Reported statistics and error estimates:
Statistical evaluation of the reproduction data and length and weight of parent animals at end of the study
The length, reproduction and mortality data were inputted into Toxrat V2.10 statistical using the OECD 211 template and the complete reports were added to the raw data. For the reproduction endpoint juveniles from living adults were calculated as no dose response effect was observed for adult mortality.

Number of living juveniles per parent animal at each nominal test concentration, mean number of neonates per concentration and coefficient of variance.

 

Reproduction

Daphnia No.

0 mg/L

0.48 mg/L

1.53 mg/L

4.88 mg/L

15.3 mg/L

50.0 mg/L

1

169

21 M

203

109 M

193

29 M

2

207

200

122 M

197

183

158

3

195

88 M

170

125

104

165

4

168

144

191

186

133

129

5

117 M

124

 M

210

126

131

6

142

125

178

195

130

161

7

154

144

156

196

119

52

8

162

147

201

146

158

150

9

186

164

164

172

133

105

10

180

59 M

138

144

173

155

Total

1680

1216

1523

1680

1452

1235

Mean

168

122

169

168

145

124

Stdev

26

52

28

35

30

48

CV%

16

43

16

21

20

39

 

M=Mortality parent animal

Validity criteria fulfilled:
yes
Conclusions:
The study met the biological and analytical validity criteria for a valid test and is expected to be a valid representation of the effects of the nominal concentrations of the test material to the test organism in fresh water. Chemical analysis initially allowed the fresh solutions to be quantified but at considerably lower recoveries than expected. Old solutions were for the majority not detected above the limit of quantification. The additional data generated in the first additional test demonstrated that recoveries could be significantly increased by extracting and analyzing the algae and water components separately. The test material is known to adsorb strongly and the initial analytical method did not adequately take into account this tendency. Adsorbed algae, is still ingested by the test organism and is still present in the test system and is a relevant route for the material to cause any potential toxicity. For this reason an alternative method at the determined reproduction NOEC was conducted to determine the total amount of test substance still present in the test system. Additional analytics from the second additional test demonstrated that a greater recovery could be achieved. For this reason it is considered justified to use this data for expression of the reproduction NOEC. Furthermore the conclusion (NOEC reproduction 1.6 mg/L) and concentrations measured with the second analytical are better in line with the historical data of the analog of the material tested and therefore better support more consistent read across of the two materials.
Executive summary:

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).

 

The primary test criterion used to indicate the toxicity of the test substance was reproductive capacity expressed as the total number of neonates per surviving parent animal at the end of the study due to there being no test substance related parent mortality. Data on parental length and dry-weight was also generated for use in determining secondary endpoints as required.

 

The nominal concentrations used in the study were as follows:

 

0.48, 1.53, 4.88, 15.3 and 50 mg/L including a test medium only control.

 

All concentrations given refer to the technical product as supplied by the sponsor. 

 

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 168 after 21 days (minimum acceptable = 60).

•          Analytical quality criteria considered needed for a robust analysis were met. 

 

Reproduction (Primary endpoint)

A NOEC and LOEC for reproduction for the substance as nominal test material were determined as 4.9 and 15.6 mg/L respectively. The EC10for reproduction was calculated as 4.1 mg/L. (Nominal concentrations) The NOEC for reproduction can also be expressed as 1.6 mg/L (Time weighted measured mean).

 

The test material may be concluded not to be toxic to the reproduction of D.Magna based on data from the primary endpoint and additional analysis to recover the test material.

Description of key information

One OECD 211 GLP test is available with an overall NOEC of 2.7 mg/L (mean measured concentration).

Key value for chemical safety assessment

EC10, LC10 or NOEC for freshwater invertebrates:
2.7 mg/L

Additional information

One OECD 211 GLP test with 3,6,9-Triethyl-3,6,9-trimethyl-1,4,7-triperoxonane (CAS 24748 -23 -0) is available with an overall NOEC of 2.7 mg/L (mean measured concentration).

This result is supported by an OECD 211 study according to protocol but without GLP. The NOEC for reproduction of this study is 1.6 mg/L (measured TWA concentration).