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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:
key study
Study period:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to guideline
OECD Guideline 211 (Daphnia magna Reproduction Test)
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
Details on sampling:
The concentration of the test substance was determined in one representative test interval per week in each freshly prepared test solution (test vessels without daphnids) and in the 48 h or 72 h old test solutions before renewal (combined sample from test vessels with daphnids).
Additionally, retained samples are collected from the 48 hour or 72 hour old abiotic replicate (no Daphnia or food algae) per test group. These samples were analyzed to assist in the interpretation of results in the event that food algae or Daphnia over the exposure period interfered with the test substance analyses.
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test concentrations were prepared by dilution of a stock solution. The stock solution (2.2 mg/L) was prepared by pipetting 11 mg (9.95 μL based on the density) test substance into 5 L of test medium. The mixture was sonicated in an ultrasonic bath for 10 minutes followed by stirring for approximately 10 min. Before the dilution the stock solution was checked for complete dissolution of the test substance. All test solutions were visibly colorless and clear throughout each renewal period.
One additional abiotic replicate (no Daphnia or food algae) per test group was used to help assess test substance stability over the renewal period.
The stability of the test substance in the test media at room temperature over a period of 96 hours was demonstrated in a preliminary investigation (

Test organisms (species):
Daphnia magna
Details on test organisms:
- Strain/clone: Daphnia magna STRAUS
- Source: The clone of Daphnia magna STRAUS 1820 used was supplied by the Institut National de Recherche Chimique Appliquée, France, in 1978. From this date on this clone was cultured and bred continuously in the current test laboratory.
- Age of parental stock (mean and range, SD): <24 h

Daphnia brood stock are kept in mass cultures consisting of approx. 20–30 individuals for a maximum of 4 weeks. All individuals in the mass culture originate from a single female. After approximately 14 days the adults have produced at least 3 broods and the young can be used in tests. Offspring are removed from the mass cultures at least once daily during the normal work week to ensure that young daphnia are <24-h old (first instar) at test initiation. Detailed records are kept (in test facility archives) to monitor the health of Daphnia brood stock cultures including observations of young production, mortality, ephippia, and measurement of water chemistry parameters.
Only young from healthy cultures without signs of stress are used for testing.

- Acclimation conditions (same as test or not): The Daphnia are cultured under the identical conditions as the test including test media (Elendt M4), water quality, temperature (20 ±1°C), and diet.

During the test daphnids were fed daily a diet of live green algae Desmodesmus subspicatus, cultured in a synthetic medium. The algae were separated from their culture medium by centrifugation, resuspended in daphnid's medium (M4) corresponding to concentrations of 121 mg and 126 mg TOC*/L (respectively) in the algal concentrates used. The daphnids were fed a defined volume (≤126 μL) of the concentrate to reach the amount of food defined in the table below. The algae were stored in a refrigerator (dark, about 4- 8°C) for maximum 21 days.
By adding the algal concentrate the test solution was slightly diluted. During a 24 h test interval with feeding a total of 0.126 mL are added to 50 mL test volume resulting in a maximal dilution of 0.25%.
Test type:
Water media type:
Limit test:
Total exposure duration:
21 d
2.39-2.53 mmol
Test temperature:
Dissolved oxygen:
8.2-9.4 mg/L
Nominal and measured concentrations:
0 (control), 0.21, 0.37, 0.67, 1.2, 2.2 mg/L as nominal concentrations based on test substance mass not corrected for purity.
0 (control), 0.157, 0.269, 0.483, 0.85, 1.78 mg/L as timeweighed mean (TWM) measured concentrations of abiotic samples based on test substance mass not corrected for purity.
Details on test conditions:
- Test vessel: Numbered glass beakers (nominal volume 100 mL), covered with glass Petri plates to slow evaporation. Test volume 50mL
- Aeration: none
- Renewal rate of test solution (frequency/flow rate): 3 times per week
- No. of organisms per vessel: 1 animal / test vessel (0.02 animals / mL)
- No. of vessels per concentration (replicates): 10, 1 parent animal each
- No. of vessels per control (replicates): 10, 1 parent animal each

A synthetic fresh water (Elendt M4) is used as media for culture and test purposes. For the composition of this M4 medium see OECD 211. The general properties of this
medium are as follows:
- Total hardness: 2.20–3.20 mmol/L
- Acid capacity up to pH 4.3: 0.80–1.00 mmol/L
- Molar ratio Ca:Mg: about 4:1
- pH value: 7.5–8.5
- Conductivity: 550–650 μS/cm
- Total organic carbon: <2 mg/L
- Dissolved oxygen: Must remain ≥3mg/L during the test. To assure optimal dissolved oxygen levels, the M4 medium is aerated for approximately 24 h prior to use.

- Adjustment of pH:
- Light intensity/photoperiod: 742–823 lux at a wave length of 400–750 nm. The light intensity will not exceed 1000–1500 lux for cool white light (equivalent to 15–20 μE•m-2•s-1 according to the test guideline. 16 hours light : 8 hours darkness

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Parent mortality, abnormal effects, and numbers of live and dead offspring: every day
- Reproductive success was measured by counting and discarding the offspring produced by each parent: for the duration of the study
- body length of each adult daphnid: test termination
- appearance of the test solutions and dissolution behavior of the test substance: daily

- chemical and physical parameters of the test medium (total hardness, acid capacity, pH, conductivity and total organic carbon): after aeration and prior to use
- Dissolved oxygen, pH and temperature were measured in the old and in the freshly prepared test solution in replicate 1 of each test group: for one interval per week
- temperature: continuously during the whole exposure period in a separate vessel filled with water proximal to the test vessels
- Hardness was measured in the freshly prepared test solution in an additional replicate of each concentration for one interval per week and in the old test solution from the combined replicates of each test group for one interval per week.

- Spacing factor for test concentrations: 1.8
- Range finding study: A preliminary range finding test was performed (experimental conduct in accordance with GLP but without a GLP status), with exposure over 17 days at the following nominal concentrations: 0 (control), 0.32, 1, 3.2 mg/L. After 17 days exposure to the test substance, statistically significant effects on reproduction were observed at 1 mg/L and 3.2 mg/L.
Reference substance (positive control):
monthly conducted with sodium chloride
Key result
21 d
Dose descriptor:
Effect conc.:
0.196 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
time weighted mean concentrations
Basis for effect:
Remarks on result:
other: 95% confidence intervals
Details on results:
A significant effect on parent mortality (Fisher’s exact test) was observed during the 21 day exposure in the highest test group (1.78 mg/L TWM, abiotic, corresponding to 2.2 mg/L, nominal). In this group mortality was first observed on day 3 and all Daphnia were dead by day 7. A single mortality was observed in test group 4, although this was not statistically significant. In order to determine if this mortality was inadvertent or substance related the data were evaluated for a concentration response pattern using a step-down Cochran-Armitage trend test as indicated in the OECD 211 test guideline. The mortality data with test group 5 was significant; however the data with test group 4 did not follow a concentration response pattern. Therefore the mortality in group 4 is considered inadvertent and the mortality in test group 5 was substance related.

Reproduction was significantly affected in all treatment groups. Since the mortality in test group 4 was inadvertent and there was no reproduction in test group 5, the reproduction data
are reported based on surviving adult as recommended in the OECD 211 guideline. Although there was a statistically significant effect on reproduction in test groups 1 and 2, the
exceptionally high reproduction rate in concurrent control should be considered. The laboratory historical control data for this species in an OECD 211 study (n=81 studies) demonstrate that mean living young per surviving adult is 136 (sd = 32; data archived with the raw data from this study).

Other observations:
Significant effects on growth were observed at ≥ 0.67 mg/L and this corresponds to a significant effect on the reproduction endpoint in these groups.
In test group 0.67 mg/L (nominal), replicate 1, the daphnid was observed to be pale and small on study day 8 and 9. In test group 1.2 mg/L (nominal) from study day 8 until study day
20 daphnids were observed to be pale and/or small in all test replicates. The daphnid in replicate 10 of this test group was dead on study day 18.
In test group 1 (0.21 mg/L, nominal) a statistically significant effect on %immobile young was observed. This effect is considered to be transient as the daphnids in all higher test
concentrations did not show a statistically significant effect.
All test solutions were visibly colorless and clear throughout each renewal period. No undissolved test substance or other unusual manifestations were observed.

This test was fully compliant with all the following validity criteria required by the corresponding test guidelines and is considered valid.
• In the control the mortality of parent animals up to the end of the test should be ≤20%.
• In the control the mean number of living young per parent animal that survived the test should be ≥60.
Results with reference substance (positive control):
The EC50(48 h) of the reference substance sodium chloride (NaCl) was 4.66 g/L. (experiment date: 07 Apr 2015)
This result is within the range of 3.88–7.22 g/L, which represents ±2 standard deviations from the published EC50(48 h) of 5.55 g/L and indicates that the culture of Daphnia magna used in this study is responding normally to toxic stress.

The mean analytically measured concentrations of the test substance in the test water were within the range of 85-93% of the nominal concentration at the start of the renewal period (initial concentrations) and decreased to 41-71 % of the measured initial concentrations in aged biotic samples at the end of the renewal period.

The concentration loss in the abiotic samples was less than in the biotic samples and ranged from 79-89% of the mean measured initial concentrations. The greater recovery in the abiotic samples indicates that loss in the biotic samples was likely due in part to adsorption to food algae.

Since test substance adsorbed to food algae is still bioavailable to the Daphnia, the time weighted mean measured concentrations using the abiotic values are considered an accurate representation of exposure concentrations maintained during the test.

Therefore, the effect concentration, which is based on the time-weighted mean measured concentrations, should be preferably used for the evaluation of the test substance.

               21 day Effect Concentrations [mg/L], TWM, abiotic
  Endpoint  NOEC  LOEC









 Reproduction  < 0.157  0.157


0.15 -0.26


0.20 -0.31



0.64 -0.86

 Mortality  0.483  0.483  0.85*  -  0.88*  0.95*

* The data were not sufficient to calculate 95% confidence intervals for mortality.

-: not calculated

               21 day Effect Concentrations [mg/L], nominal
  Endpoint  NOEC  LOEC









 Reproduction  < 0.21  0.21


0.20 -0.35


0.28 -0.43



0.89 -1.22

 Mortality  1.2  2.2  1.2*  -  1.24*  1.32*

* The data were not sufficient to calculate 95% confidence intervals for mortality.

-: not calculated

Description of key information

Toxic effects were observed in a Daphnia magna reproduction test according to OECD 211.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Dose descriptor:
Effect concentration:
0.196 mg/L

Additional information

The long-term toxicity on aquatic invertebrates was investigated with Daphnia magna, in a GLP guideline study according to OECD 211 with semi-static exposure. 

Following concentrations were tested: 0 (control), 0.21, 0.37, 0.67, 1.2, 2.2 mg/L as nominal.

A significant effect on parent mortality was observed during the 21 day exposure in the highest test group. Reproduction was significantly affected in all treatment groups.

Since the measured concentrations deviated markedly from the nominal concentrations, the effect concentration is based on the time-weighted mean measured (TWM) concentrations:

EC10 reproduction 21 day : 0.196 mg/L (TWM)