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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:
05 Dec 2016 to 03 Apr 2017
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to
OECD Guideline 211 (Daphnia magna Reproduction Test)
Version / remarks:
October 2012
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
- Name of test material (as cited in study report): zinc bis(dibenzyldithiocarbamate)
- Batch No.: 50705100
- Purity: 98.7%
- Date of receipt: 19 October 2015
- Expiration date: 31 July 2017
- Appearance: withe to light cream powder
Analytical monitoring:
Details on sampling:
Samples for analysis were taken from all test concentrations and the control on 4 occasions throughout the test, on fresh and old solutions:
- at days 0 (fresh solutions) and 1 (old solutions)
- at days 10 (fresh solutions) and 11 (old solutions)
- at days 17 (fresh solutions) and 18 (old solutions)
- at days 20 (fresh solutions) and 21 (old solutions)
Samples were analysed on the same day as sampling.
Details on test solutions:
Tests solutions were prepared daily from dilutions of a saturated stock solution prepared at 1 mg/L of nominal concentration: a known volume of test item (around 2 mg for the preliminary range-finding test and 3 mg for the definitive test), was poured into a volumetric flask (2L and 3L for the definitive test,), the volume was then made up with test medium. The solution was kept under high speed stirring at ambient temperature during approximately 24h hours with a magnetic stir bar. Due to the presence of undissolved particles, solutions were filtered through filters (0.22 μm for the preliminary range-finding test and 0.45 μm for the definitive test) before dilution.
Test organisms (species):
Daphnia magna
Details on test organisms:
- Common name: Daphnid
- Clone: 5
- Age: The neonates used were less than 24h old at test initiation (selected by filtration) and were not first brood progeny.
- Feeding during test: Yes. Test animals were fed a diet of 0.1 - 0.2 mg of carbon per daphnid per day, in the form of a suspension of the algal strains Chlorella vulgaris (25%) and Pseudokirchneriella subcapitata (75%), except during the initial three days of culture when a slightly lower ration was given. The concentrated algal cell suspension was prepared by removing and centrifuging aliquots of algal culture and resuspending the algal pellets in small volumes of dilution medium.
Test type:
Water media type:
Limit test:
Total exposure duration:
21 d
- Control: 150 - 160 mg/L as CaCO3
- 100.0%: 190 - 200 mg/L as CaCO3
Test temperature:
20.0 - 20.6 °C
7.5 - 8.5
Dissolved oxygen:
8.2-9.8 mg O2/L
Nominal and measured concentrations:
- Saturated solution concentrations: 0 (control), 12.0, 20.4, 34.6, 58.8 and 100% v/v saturated test susbtance solution
- Mean (geometric) measured concentrations:
Details on test conditions:
- Test vessel: Test vessels were made of clear glass tubes (120 mL capacity) to minimise evaporation and avoid the entry of dust into the solutions
- Type: Closed; the test vessel was stoppered with cellulose bungs
- Fill volume: 50 mL
- Renewal rate of test solution: All test solutions (including control) were renewed every day (including weekends).
- No. of organisms per vessel: 1
- No. of vessels per concentration: 10
- No. of vessels per control: 10

- Source/preparation of dilution water: All testing was performed using a synthetic medium prepared with Volvic® water and complemented as follow:
- 0.1 ml/L of B12 vitamin solution (1 μg/L)
- 0.1 ml/L of Na2SeO3.5H2O solution (6.7 μg/L)
- 1 ml/L of calcium / magnesium solution: Ca(NO3)2.4H2O : 208 g/L and MgCl2.6H2O : 28 g/L.
Chemical reagents used for the preparation of this dilution water were of “analytical grade”.
- Culture medium different from test medium: Same

- Temperature: The temperature was continuously recorded throughout the test in a flask maintained within the same conditions.
- pH and dissolved oxygen: pH and dissolved oxygen were measured in the fresh and old solutions in one of the vessels of each
test group with surviving daphnids.
- Hardness: The total hardness used to prepare test solutions was determined using a Hardness test kit (MColortest®, Merck).

- Photoperiod: light cycle of 16 h light and 8 h dark was in operation throughout the test
- Light quality: Light was provided by LED (light spectrum of 400 - 700 nm, Lumen 3450 lm)
- Light intensity: The light intensity was measured between 650 - 1200 lux.

EFFECT PARAMETERS MEASURED: immobility, reproductive effects
Every day, all parent animals were observed for immobilisation by gentle agitation of the test vessel. Any immobile animal within 15 seconds following agitation was considered as dead and discarded. The surviving parental Daphnia were transferred into fresh control or test media in a second set of vessels by means of a wide bore glass pipette. From the day of the first brood, all neonates (dead and alive), unhatched eggs or carapaces present in the expired media were counted daily and discarded. Any other abnormal observation was also recorded and reported.

The preliminary test was conducted at the nominal test item concentrations of 12.5, 25.0, 50.0 and 100.0% of the saturated solution, together with a control group. Five replicate vessels were prepared at each test concentration (except for the test concentration 100% for which one organism was omitted in a replicate). Each vessel contained 1 daphnid and algae (0.1-0.2 mg C). The total test period was 10 days. All test solutions (including control) were renewed every day (including weekends). Samples for analysis were taken from all test concentrations and the control on 2 occasions throughout the test, on fresh and old solutions at days 0 (fresh solutions) and 1 (old solutions); AND at days 9 (fresh solutions) and 10 (old solutions).
- Results used to determine the conditions for the definitive study: No immobile adult Daphnia magna was recorded throughout the test at each concentration.
Offspring were observed in all group by Day 9. These results indicates that there was no effect on D. magna reproduction at any concentration tested.
Reference substance (positive control):
The sensitivity of the test system and the methodology are evaluated monthly by performing an acute toxicity test on potassium dichromate.
Key result
21 d
Dose descriptor:
Effect conc.:
>= 16.9 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat. (dissolved fraction)
corresponding to 100% saturated solution
Basis for effect:
Remarks on result:
other: No effects observed. The NOEC was determined as greater than the maximum solubility of test item.
Details on results:
An overview of the results is presented in 'Any other information on results incl. tables'.

- Test substnace appearance in solution: All test solutions appeared clear and colourless. No precipitation of test item nor undissolved particles were observed.
- Immobility: The number of immobile adult D. magna recorded during the test were 1 (10%) at 3.3, 6.2 and 11.5 μg/L. The immobile adult D. magna at 11.5 μg/L was recorded by Day 2 and considered as accidental mortality. No immobile D. magna were recorded at 1.7 and 16.9 μg/L, or in the control. The lack of significant adult immobilisation in the test vessels compared with the control indicated that the test item has no effect on D. magna survival over a 21 day period, under test conditions.
- Reproduction inhibition: A statistical analysis of complete data shown significant effect on Daphnia reproduction at the test solution 11.5 μg/L (21d-LOEC = 11.5 μg/L). However the lack of a reduction in offspring produced at the highest tested concentration of 16.9 μg/L suggests that the test item is not involved. The sub lethal effects observed on organisms from Days 14/15 at the test concentration 11.5 μg/L as well as the lack of a reduction in offspring produced at all tested concentration until Day 14 reinforced this hypothesis. The data obtained at the test solution 11.5 μg/L were thus disregarded from the statistical analysis.
- Age at first reproduction: Offspring were observed in the 1.7, 6.2 and 11.5 μg/L treated group by Day 8, with offspring present in all other groups by Day 9. This difference was statistically considered as non-significant. The test item has thus no effect on D. magna reproduction over a 21 day period.
Results with reference substance (positive control):
The latest value of 24h-EC50 (obtained on January 2017) was 0.96 mg/L.
Reported statistics and error estimates:
The geometric means of the concentrations measured in new and old media over 24-h periods were used for the estimation of ECx and NOEC/LOEC values. Statistical analysis was conducted on the following variables: number of living neonates produced by surviving parent, parent mobility and age at first brood. All statistical analysis were conducted using the Software ToxRat 2.10.

Table: Summary of the results


Test solution (% saturation / retained concentration in µg/L)


12.0 / 1.7

20.4 / 3.3

34.6 / 6.2

58.8 / 11.5

100.0 / 16.9

Total of living

offspring (per parent














Mean of living

offspring produced

per parent













Standard Deviation







Total of dead offspring







Total of eggs







Total of dead parent







*Produced at the end of the test by all the parent daphnia integrated at the start of the test excluding parental accidental and/or inadvertent mortality

**Accidental mortality: the organism was found to be stuck to the test vessel on Day 2

Validity criteria fulfilled:
See 'Any other information on materials and methods incl. tables'.

Description of key information

No effects up to the water solubility limit were observed in a study according to OECD TG 211 and in compliance with GLP criteria. The 21-day NOEC was therefore determined to be greater than or equal to 19.6 µg/L for Daphnia magna exposed to the test substance.

Key value for chemical safety assessment

EC10, LC10 or NOEC for freshwater invertebrates:
19.6 µg/L

Additional information

The long-term toxicity to aquatic invertebrates was determined in a study according to OECD 211 and in compliance with GLP criteria (Arkema, 2017). In this study daphnids (D. magna, 10 per concentration) were exposed to saturated solutions of the test substance of 0, (control), 12.0, 20.4, 34.6, 58.8 and 100% for 21 days under semi-static conditions. The test solutions (including control) were renewed daily throughout the test.Test concentrations were analytically verified at each renewal (old and new) and were determined to be<LOQ, 1.7, 3.3, 6.2, 11.5 and 16.9 µg/L (geometric mean). Immobility of the parent organisms was observed daily. The number of juveniles produced per parental animal on any day during the 21-day study duration was recorded.

In the 3.3, 6.2 and 11.5 μg/L treatments, one daphnid was found immobile throughout the test. No immobile daphnia were observed in the control, lowest and highest test concentration treatments. Furtheremore, no significant relevant effects were observed on reproduction inhibition and age at first reproduction. The findings indicate that the test substance has no effect on survival and reproduction over a 21 day period under the test conditions chosen. Therefore, the NOEC was determined to be greater than the maximum solubility of the test item.