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Long-term toxicity to aquatic invertebrates

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long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
supporting study
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
equivalent or similar to guideline
OECD Guideline 211 (Daphnia magna Reproduction Test)
14d range finding test with chemical analyses
GLP compliance:
Analytical monitoring:
Details on sampling:
Samples of all new test concentrations and old stock solutions were taken as often as possible in the study. New samples were taken in the test vessels before the addition of algae. Samples were subsequently transferred to HPLC vials. Old samples were taken from pooled test vessels and filtered prior to the addition to HPLC vials. Samples were diluted in test medium to ensure that they remained in the range of the calibration curve.
Details on test solutions:
Preparation of the stock solutions:
The test substance is dissolvable at the required test concentrations. To prepare the stock solutions for every medium renewal, between 0.2003 and 0.2013g of test substance was weighed on an analytical balance. The test substance was transferred to a 1L volumetric flask using a watch glass and glass funnel. The entire 1 L of test medium was used to rinse the watch glass and funnel in order to make a stock as close to the nominal concentrations as possible. Although soluble the stock required stirring for at least one hour before a homogeneous stock was achieved. The stock pH in the first stock was found to be pH 8 and therefore none of the stock solution made during the study were pH adjusted due to the pH being close to that of the test medium.

Preparation of the test solutions:
Test solutions were prepared by further dilution of the relevant stock solutions with test medium in volumetric flasks. All pipetting took place while stock was under agitation and all pipettes were rinsed at least once with the stock and the contents discarded. Test vessels were filled directly from volumetric flasks immediately after preparation. The solutions were renewed 5 times a week. A new stock was not always made.
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. The most recent reference test results were within the range given in the guideline and the culture was deemed suitable for use. In addition no abnormal mortality was observed in the cultures and no winter eggs were present.

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:
Water media type:
Limit test:
Total exposure duration:
14 d
Test temperature:
19.6-20.6 degree C
Dissolved oxygen:
8.7 - 10.3 mg O2/L
Nominal and measured concentrations:
Nominal: 0.48, 1.52, 4.88 15.6 and 50 mg/L
Time weighted average measured concentrations: 0.16, 0.54, 1.66, 6.29 and 16.8 mg/L
Details on test conditions:
- Test vessel: 100 mL glass beakers
- Type (delete if not applicable): open
- Aeration: No
- Renewal rate of test solution (frequency/flow rate): daily renewal, 5 times per week
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10

- Source/preparation of dilution water: M4 Elendt medium
- Culture medium different from test medium: No

- Adjustment of pH: no
- Photoperiod: 16h light

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : number of offspring, parent mortality, length and weight

- Spacing factor for test concentrations: 3.2
Reference substance (positive control):
not specified
Key result
14 d
Dose descriptor:
Effect conc.:
0.1 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
14 d
Dose descriptor:
Effect conc.:
1.1 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
14 d
Dose descriptor:
Effect conc.:
0.16 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
Details on results:
Parent animal mortality:
One parent animal died at 4.88 mg/L and all parent animals died at the highest concentration.

Coefficient of variation of control fecundity:
A high level of variation was observed in the reproduction of the control. This is also represented by the high number of immobile juveniles observed. The study director has observed that daily refreshment in synthetic medium has also been observed to cause negative effects to reproduction behavior in
previous studies. Possibly due to reduced food or due to regular disturbance of the test animals.
However this method was chosen to maximize test substance exposure. The daphnia culture was otherwise healthy.

Reproduction (Primary endpoint):
The EC10 and EC50 for reproduction were calculated as 0.1mg/L and 1.1 mg/L respectively based on time weighted mean concentrations. The NOEC that was determined statistically (1.6mg/L) was not considered reliable due to the excessive control variation making the determination two broad and therefore not sufficiently worst case. For this reason the study director has set the NOEC at the first test concentration in which equal or more juveniles were observed when compared to the control. The NOEC was therefore determined as 0.16 mg/L based on time weighted mean concentrations.

Length (Secondary endpoint):
No significant difference in length was detected in all surviving adults

Dry weight (Supporting endpoint):
No significant difference in dry weight was visible in the dry weight data.

EC50 for parent animals:
An EC50 based on survival of parent animals at the end of the test was determined as 9.4mg/L. The NOEC was determined as 6.3 mg/L based on time weighted mean concentrations.

Any other biological effects observed:
A large numbers of juveniles were born early at all concentrations including the control and were scored immobile / dead. In reality many of these juveniles were alive but distinguishing between them was not always possible. These results were emitted from statistical analysis and only the clearly alive and mobile juveniles were used for endpoint determination. The nature of the daily refreshment can result in less food in the early stages of the study and/or more disruption to the parent animals. However a test substance related effect was observed on the living juveniles and the control animals were otherwise in good health.
Reported statistics and error estimates:
The data on reproduction and parental length were imputed into the Toxrat V2.10 professional statistical software package and assessed using the OECD 211 template. The OECD templates are closed and cannot be altered and are therefore considered to remain valid for the OECD 211 guideline without further validation.
Validity criteria fulfilled:
all criteria were met except acceptable control variation (25%).
Test results give a good indication for the long-term endpoint and for the definitive range for the final study.
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 based on the OECD Guideline No. 211 (OECD, 2012). The study was modified and conducted for a shorter 14 day period as a range finding test in preparation for a planned definitive GLP study.

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 their being no dose response related parent mortality where reproduction took place. 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, 1.52, 4.88 15.6 and 50 mg/L including a test medium only control.

All concentrations given refer to the technical product as supplied by the sponsor. Endpoints are all based on time weighted mean concentrations.

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).

 No parent mortality occurred

 The average number of juveniles per parent animal alive at the end of the test in the control (60 in a standard test) was for this shortened test not a strict quality criterion. However considering the length of the study and applying the same criteria an average of 39 juveniles would be expected. The average number of juveniles per adult in the control in this study was 39. This criterion is considered to have been met.

 Analytical quality criteria demonstrating a suitably robust analytical method were met.

The following quality criterion was not met:

 Acceptable control variation (25%) as recommended in the test guideline was significantly exceeded as a result of the daily solution refreshment. The study director accounted for this in the endpoint calculation and applied a worst case NOEC based on living juveniles per adult at the end of the test. This is considered acceptable until a definitive GLP endpoint becomes available.

The EC10 and EC50 for reproduction were calculated as 0.1mg/L and 1.1 mg/L respectively based on time weighted mean concentrations (TWM). The NOEC for reproduction was determined manually by the study director as 0.16 mg/L (TWM). The secondary endpoints showed no significant effects or did not contain significant replicates for a reliable statistical analysis.

Description of key information

A first brood screening study has been performed. The study was not performed under GLP and can be used as range finding study for the definitive OECD 211 study.

Chemical analyses were performed. The endpoint is based on time weighted mean measured concentrations. No effects were seen up to 0.16 mg/L. The EC10 for reproduction is 0.1 mg/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
0.1 mg/L

Additional information