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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:
15 - 17 Mar 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
GLP compliance:
yes (incl. QA statement)
Remarks:
Staatliches Gewerbeaufsichtsamt, Hildesheim, Germany (03 Jan 2017)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Control, 1.94, 4.27, 9.39, 20.7, 45.5, and 100% (dilution level of the saturated solution)
- Sampling method: At the start of the exposure and at renewal (0 and 24 h), samples were taken from the freshly prepared concentration levels and the control and analyzed. At renewal and at the end of exposure (24 and 48 h), samples of the 24-h old media were taken directly from the test vessels and analyzed.
- Sample storage conditions before analysis: All samples were stored at 6 ± 2 °C until sample preparation and at room temperature until the start of the analysis (on an autosampler), if necessary. Prepared extracts were stored at 6 ± 2 °C for potential reanalyses.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A saturated solution with a nominal concentration of 10 mg/L test item, which corresponds to 11.1 µL/L test item (relative density 0.9025 taken into account), was prepared two days prior to the start of the exposure. The glass flask was filled with 39.4 g glass pearls and up to the top with the dilution water having no headspace (total volume: 2275 mL). The test item was pipetted onto a glass slide and inserted into the brown glass flask and was closed immediately with a screw cap with a septum. This dispersion was shaken for 48 h with 20 rpm at room temperature and then left for 1 h to settle. The saturated solution was taken from the approximate center of the water body and checked via laser beam (Tyndall effect) for undissolved test item (formation of an emulsion). A Tyndall effect was observed in each approach. The saturated solution was used as the highest concentration level and as a stock solution for the preparation of further dilution levels by dilution with dilution water.
- Controls: Dilution water without test item incubated under the same conditions as the test groups.
- Evidence of undissolved material: All tested concentration levels were visually clear throughout the exposure period. The saturated solution had a Tyndall effect (observed after preparation at 0 and 24 h).
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: water flea
- Strain/clone: Clone 5
- Breeding conditions: The daphnia were bred at the testing facility in glass vessels (2 - 3 L capacity) with approximately 1.8 L culture medium, at 20 ± 2 °C, in an incubator, 16 h illumination, light intensity of max. 1500 lx.
- Food type: A mix of unicellular green algae, e.g. Pseudokirchneriella subcapitata and Desmodesmus subspicatus, with an algae cell density of > 10^6 cells/mL.
- Amount: Ad libitum.
- Frequency: At least 5 times per week.
- Ages of daphnia at the start of exposure: Less than 24 h old from a healthy stock.
- Feeding during test: No feeding during test

ACCLIMATION
- Acclimation period: Acclimatization was not necessary because the dilution water was equivalent to the culture medium.

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES:
Juvenile daphnia were removed from the culture vessels at the latest 24 h before the start of the exposure and discarded. The juveniles born within the following period of max. 24 h preceding the exposure were used for the test. No first brood progeny was used for the test.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
160 - 180 mg CaCO3/L
Test temperature:
19 - 20 °C
pH:
7.04 - 8.50 (fresh media)
7.99 - 8.86 (old media)
Dissolved oxygen:
8.76 - 9.88 mg O2/L (fresh media)
7.79 - 9.50 mg O2/L (old media)
Nominal and measured concentrations:
nominal concentrations: control, 1.94, 4.27, 9.39, 20.7, 45.5, and 100% of the saturated solution
geometric mean measured concentration: control, 0.153, 0.355, 0.821, 1.78, 4.12, and 9.25 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Flasks
- Type: Sealed with screw caps
- Material, size, headspace, fill volume: Material: glass; Size: 4.5 ID x 9.5 cm H; Headspace: none; Fill volume: 130 mL
- Renewal rate of test solution: The test solutions were renewed after 24 h. For this purpose, a second set of test vessels were filled up to the top with the freshly prepared test solutions and the daphnia were transferred by pipette. There was no headspace in the test vessels.
- 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: Same as culture medium (Elendt M4, according to OECD 202)
- Total water hardness: modified to 160 - 180 mg CaCO3/L (original recipe: 293.8 mg/L)
- Culture medium different from test medium: Culture medium same as test medium.
- Intervals of water quality measurement: Prior to the start of the exposure (0 h) and the renewal of the test solutions (24 h), the water quality parameters (i.e. pH-value, dissolved oxygen concentration, temperatue, conductivity and total hardness) of the dilution water were measured. At the start of the exposure and at renewal (0 and 24 h), the water quality parameters of the fresh media (i.e. pH-value, dissolved oxygen concentration) were measured in one additional replicate (without daphnia) per concentration level and control. At renewal and at the end of the exposure (24 and 48 h), the water quality parameters of the old media were measured in all replicates (containing daphnia) per concentration level and control.

OTHER TEST CONDITIONS
- Photoperiod: 16 h ligh/ 8 h dark cycle
- Light intensity: Diffuse light, max 1500 lx

EFFECT PARAMETERS MEASURED:
- Immobilization: at 24 and 48 h

RANGE-FINDING STUDY
- Test concentrations: A saturated solution of the test item at a loading rate of 10 mg/L (corresponding to 11.1 µL/L) and two further dilution levels prepared by diluting the saturated solution by a factor of 10 and 100 with dilution water.
- Results used to determine the conditions for the definitive study: Yes, the test concentrations were selected based on the results of the non-GLP preliminary range finding test: 100% immobilization after 24 h in treatments with the saturated solution (100%)
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
1.34 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% confidence interval: 1.01 - 1.72 mg/L
Details on results:
- Other biological observations: No
- Mortality of control: 0%
- Any observations that might cause a difference between measured and nominal values: All tested concentration levels were visually clear throughout the exposure period. The saturated solution had a Tyndall effect (observed after preparation at 0 and 24 h).
- Effect concentrations exceeding solubility of substance in test medium: No
Results with reference substance (positive control):
- Results with reference substance valid? Yes, within 0.6 - 2.1 mg/L, according to OECD 202 (clone A) and 0.6 - 2.4 mg/L, according to AQS P 9/2 (clone 5).
- Relevant effect levels: EC50 (24 h) = 2.05 mg/L potassium dichromate (95% confidence limits: 2.00 - 3.71 mg/L).
- Other: Exposure phase: 09 - 10 Mar 2017
Reported statistics and error estimates:
The EC10 and EC50 values after 24 and 48 h of exposure were calculated by sigmoidal dose-response regression with the software GraphPad Prism. The respective 95% confidence limits were calculated from the standard error and the t-distribution. All calculations were carried out from the best-fit values with the soft-ware GraphPad Prism.

ANALYTICAL RESULTS

Table 1. Measured concentrations of the test item during the definitive test.

Sampling date

0 h

Start of the exposure interval

24 h end of the exposure interval

24 h start of the exposure interval

48 h end of the exposure interval

 

 

 

 

Geometric mean measured test item concentration

[mg/L]

Dilution level of the saturated solution [%}

 

Measured conc.

[mg/L]

 

Meas. conc.

[mg/L]

 

%

 

Measured conc.

[mg/L]

 

Meas. conc.

[mg/L]

 

%

100*

9.78

8.74

89

Not measured due to 100% mortality after 24 h

9.25

45.5

4.47

3.97

89

4.26

3.81

90

4.12

20.7

1.96

1.64

83

1.85

1.69

91

1.78

9.39

0.889

0.831

93

0.845

0.7271)

86

0.821

4.27

0.422

0.330

78

0.387

0.2962)

76

0.355

1.94

0.165

0.137

83

0.170

0.1442)

85

0.153

Control

< LOQ

< LOQ

< LOQ

< LOQ

 

Meas. conc.     = measured concentration of the test item, single determinations, enrichments and dilution factors taken into account

%                         = percentage of the initially measured concentration of the test item

LOQ                     = limit of quantification of the analytical method (0.01 mg test item/L)

1)                                   = due to technical reasons the samples had to be reanalyzed on 2017-02-23 (after the expiry date 2017-03-17)

2)                          = Reinjection on 2017-03—23 (sample prepared on 2017-03-17)

*                          = saturated solution

 

 

BIOLOGICAL RESULTS

 

Table 2. Immobilization rates after 24 h and 48 h of exposure (n = 20, in 4 replicates).

 

Geometric mean measured concentration of the test item [mg/L]

 

IMMOBILIZATION [%]

24 h

48 h

Mean value in absolute numbers

Mean value in %

Mean value in absolute numbers

Mean value in %

9.25

20/20

100

20/20

100% after 24 h

4.12

17/20

85

20/20

100

1.78

10/20

50

13/20

65

0.821

0/20

0

5/20

25

0.355

0/20

0

4/20

20

0.153

0/20

0

0/20

0

Control

0/20

0

0/20

0

 

 

Validity criteria fulfilled:
yes
Conclusions:
An EC50 (48) of 1.34 mg/L (geometric mean measured) was obtained for the short-term toxicity to aquatic invertebrates.
Executive summary:

The effects of the test item to aquatic invertebrates were determined in an acute immobilization test according to OECD guideline 202 in a closed system without headspace under semi-static conditions (renewal: 0 and 24 h). Daphnia were exposed to five dilution levels of a saturated solution for 48 h. The concentration of the test item was analytically verified by GC-MS.

The measured concentrations of the test item in the fresh media ranged from 0.165 to 9.78 mg/L. The concentrations in the old media (24 h and 48 h) were in the range of 76 - 93% of the initially measured concentrations. The derived EC50 (48 h) was 1.34 mg/L (geometric mean measured).

Description of key information

EC50 (48 h) = 1.34 mg/L (geom. mean measured conc., D. magna, OECD 202)

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
1.34 mg/L

Additional information

There is one study available, in which the toxicity of the substance to aquatic invertebrates was investigated according to OECD guideline 202 and GLP.

In a semi-static test, Daphnia magna was exposed to an undiluted saturated solution with a nominal loading of 10 mg test item/L and five dilution levels prepared from the saturated solution in a geometric series with a dilution factor of 2.2 (nominal: 1.94 – 45.5%) for 48 h. To reduce contact with air and losses of the test item by evaporation, the test was conducted in a closed system without headspace. The concentrations of the test item were analytically verified by GC-MS in fresh media (0 and 24 h) and aged media (24 and 48 h) in all concentration levels and the control.

The measured concentrations of the test item in fresh media (at 0 and 24 h) ranged from 0.165 to 9.78 mg/L. The measured concentrations in the aged media (at 24 and 48 h) were 76 – 93% of the initially measured concentrations. The geometric mean measured concentrations were: 0.153, 0.355, 0.821, 1.78, 4.12, 9.25 mg/L.

After 48 h, immobility was observed at concentration levels above 0.153 mg/L. The derived EC50 (48 h) is 1.34 mg/L (geometric mean measured).