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Short-term toxicity to aquatic invertebrates

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Reference
Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09 January 2017 - 25 January 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
2004
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: OECD Series on Testing and Assessment, No. 23, "Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures"
Version / remarks:
December 15, 2000
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: control and all test concentrations
- Sampling method: 20 mL were taken from the freshly prepared test media of all test concentrations and the control at the start of the test, and the test medium renewal at Day 1; 20 mL were collected from pooled test media of all test concentrations and the control at the end of the first renewal period at Day 1, and at the end of the test (t=48 h)
- Sample storage conditions before analysis: All samples were extracted stand-by with hexane and the extracts were stored in a refrigerator at 4°C ± 4°C until analysis.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: preparation of a saturated solution (direct addtion to test medium) - Due to the low limit of water solubility and oily characteristic of the test item a water accommodated fraction (WAF) was prepared. Additionally, the test item is highly volatile. Therefore a supersaturated stock solution of only the highest concentration (330 mg/L) was prepared to dissolve as much substance as possible. The stock solution was stirred gently for 24 hours. Vessels with a conical glass stopper were used and the head space was reduced to a minimum to minimise volatility. After the stirring period, the stock solution was allowed to rest for 1 hour until phase separation. Then, the test medium was extracted from below the water surface and used as the highest test concentration. The lower concentrations were prepared by diluting the highest concentration with adequate volumes of test water to reduce loss due to volatilisation. The test media were prepared just before introduction of the daphnids (= start of the test) and test medium renewal on Day 1.
- Controls: test medium without test substance
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): yes, in the freshly prepared highest test concentration (= 100% saturated solution) some test item was floating at the water surface as small droplets
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna (Straus)
- Strain/clone: clone 5
- Source: test facility's in-house laboratory culture
- Feeding during culture:
* Food type: green algae (Desmodesmus subspicatus)
* Frequency: daily
- Feeding during test: no
- Age at study initiation: young daphnids with an age of < 24 hours

ACCLIMATION
- Acclimation period: not necessary, since the test was performed in the same medium as the culturing

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES:
The daphnids were bred in the laboratories of ibacon under similar temperature and light conditions as used in the test. The cultivation of the parental daphnids was performed in Elendt M4 medium. The test organisms were not first brood progeny.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Post exposure observation period:
no
Hardness:
250 mg/L (as CaCO3)
Test temperature:
19.3-20.0°C
pH:
fresh solutions: 8.0-8.1
used solutions: 8.0
Dissolved oxygen:
fresh solutions: 8.7-9.0 mg O2/L
used solutions: 8.3-8.8 mg O2/L
Conductivity:
< 5 μScm^-1
Nominal and measured concentrations:
Due to the low limit of water solubility and volatility a water accommodated fraction (WAF) was prepared with a nominal loading rate of 300 mg test item/L as highest test concentration. Dilutions of the highest concentrations of 1:2, 1:4, 1:8 and 1:16 and a control were tested.

The WAF and its dilutions are corresponding to the following geometric mean measured concentrations of the test item:
0.141, 0.059, 0.029, 0.014 and 0.008 mg test item/L

Effect parameters are beased on geometric mean measured concentrations since the test item concentrations were not within ± 20% of the measured initial concentrations during the test. Additionally, preparation of fresh test solutions proved not reproducible (at renewal only 57.6-69.2% of the concentrations measured at t=0 were achieved; calculated by the reviewer).
Details on test conditions:
TEST SYSTEM
- Test vessel: Glass beakers of approximately 110 mL volume containing as much test medium as possible (i.e. the remaining head space was reduced to a technical possible minimum of some mL), kept closed during the whole period of the study with a conical glass stopper to avoid loss of the test item due to volatilisation.
- Aeration: no
- Renewal rate of test solution (frequency): 1 renewal after 24 hours
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Elendt M4 according to OECD202
- Alkalinity: 0.9 mmol
- Culture medium different from test medium: no
- Intervals of water quality measurement: temperature, pH-values and dissolved oxygen concentrations were determined in all freshly prepared and aged test media of each treatment group

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 h light : 8 h dark
- Light intensity: 440 to 720 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : mobility (at 24 and 48 hours)

RANGE-FINDING STUDY
- Test concentrations: not specified in the report
- Results used to determine the conditions for the definitive study: yes
Reference substance (positive control):
yes
Remarks:
potassium dichromate (September 2016)
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
0.119 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
mobility
Remarks on result:
other: 95% C.I. 0.086-0.216 mg/L
Details on results:
- Behavioural abnormalities: no
- Mortality of control: no
- Other adverse effects control: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: no
- Other:
* Relative to initial [%] (based on the mean of measured conc.) ranged from 29.4 - 48.5% in the first renewal period and 27.5-55.9% in the second renewal period (calculated by the reviewer)
Results with reference substance (positive control):
- Results with reference substance valid? yes
- Dose-response test: yes; tested concnetrations: 0.125, 0.25, 0.5, 1.0 and 2.0 mg/L
- ECx: 24h-EC50 = 0.959 mg/L (no 95% C.I. determinable); 48h-EC50 = 0.765 (95% C.I. 0.650-0.888 mg/L)
- Other: the sensitivity of the daphnids was consistent with the level proposed by the OECD 202 guideline (i.e. 24h-EC50 between 0.6 and 2.1 mg potassium dichromate/L)
Reported statistics and error estimates:
The EC50 and EC10 and the 95 % confidence limits were calculated by probit analysis.
The NOEC was determined directly from the raw data.
The software used to perform the statistical analysis was ToxRat Professional, Version 3.2.1, ToxRat Solutions GmbH.

Cumulative Immobilization Data and Observations in the Definitive Test

Geometric mean measured 

Concentration

[mg/L]

No. of Daphnia tested

No. of immobilized Daphnia

after

% of immobilized Daphnia after

24 h

48 h

24 h

48 h

 

 

Control

20

0

0

0

0

0.008

20

0

0

0

0

0.014

20

0

0

0

0

0.029

20

0

0

0

0

0.059

20

0

7

0

35

0.141

20

0

10

0

50

 

Summary of Analytical Results

Nominal

Test Concentration

Fresh test media

Test Concentration

Aged test media

Test Concentration

Geometric Mean

Test Concentration

 

[mg/L]1

[mg/L]2

[mg/L]3

Control

n.a.

n.a.

n.a.

WAF 1:16

0.014

0.005*

0.008

WAF 1:8

0.022

0.008*

0.014

WAF 1:4

0.043

0.020

0.029

WAF 1:2

0.088

0.039

0.059

WAF of 300 mg test item/L

0.196

0.101

0.141

1initial mean measured: mean value of all freshly prepared test media per treatment group

2mean value of all aged test media per treatment group

3geometric mean value calculated using initial mean recovery and aged mean recovery rates per treatment group

*half the Limit of Quantification (LOQ) was used for the aged test item concentrations where the values were below the LOQ, as permitted by the OECD 23 guideline

n.a.: not applicable

Validity criteria fulfilled:
yes
Remarks:
1) In the control, no daphnids became immobilised or showed other signs of disease or stress. 2) The oxygen concentration at the end of the test was ≥3 mg/L in control and test vessels.
Conclusions:
The 48h-EC50 value to Daphnia magna was 0.119 mg/L based on geometric mean measured concentrations.
Executive summary:

A study was performed to assess the acute toxicity of the substance to Daphnia magna. The study was conducted in accordance with OECD Guideline for Testing of Chemicals No. 202 and GLP under semi-static conditions. Based on the results of a range-finding test, the following nominal concentrations were tested in the definitive test: untreated control, a water accommodated fraction (WAF) prepared at a nominal loading rate of 300 mg test item/L as highest test concentration and dilutions of the highest concentrations of 1:2, 1:4, 1:8 and 1:16. For each concentration and a control group, twenty Daphnia magna (less than 24 hours old) were exposed for 48 hours in air-tight closed vessels. The incidence of immobilisation was recorded for each test and control group at 24 hours and at 48 hours. Samples taken from all concentrations at the start and the end of both 24 -hour renewal periods were analysed with a validated GC-FID method.

Measured concentrations in used test solutions ranged from 29 -49% of initial in the first renewal period, and from 26 -58% of initial in the second renewal period. Since test concentrations did not remain stable during the 24 -hour renewal periods (i.e. not within 80 -120% of initial), geometric mean measured concentrations were calculated. The range tested based on geometric mean measured concentrations was 0.141, 0.059, 0.029, 0.014 and 0.008 mg test item/L. The 48h-EC50 value to Daphnia magna was 0.119 mg/L based on geometric mean measured concentrations.

Description of key information

A study was performed to assess the acute toxicity of the substance to Daphnia magna. The study was conducted in accordance with OECD Guideline for Testing of Chemicals No. 202 and GLP under semi-static conditions. Based on the results of a range-finding test, the following nominal concentrations were tested in the definitive test: untreated control, a water accommodated fraction (WAF) prepared at a nominal loading rate of 300 mg test item/L as highest test concentration and dilutions of the highest concentrations of 1:2, 1:4, 1:8 and 1:16. For each concentration and a control group, twenty Daphnia magna (less than 24 hours old) were exposed for 48 hours in air-tight closed vessels. The incidence of immobilisation was recorded for each test and control group at 24 hours and at 48 hours. Samples taken from all concentrations at the start and the end of both 24 -hour renewal periods were analysed with a validated GC-FID method.

Measured concentrations in used test solutions ranged from 29 -49% of initial in the first renewal period, and from 26 -58% of initial in the second renewal period. Since test concentrations did not remain stable during the 24 -hour renewal periods (i.e. not within 80 -120% of initial), geometric mean measured concentrations were calculated. The range tested based on geometric mean measured concentrations was 0.141, 0.059, 0.029, 0.014 and 0.008 mg test item/L. The 48h-EC50 value to Daphnia magna was 0.119 mg/L based on geometric mean measured concentrations.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
0.119 mg/L

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