[No public or meaningful name is available]

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05 March 2018 to 22 March 2018

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

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The water-accommodated fraction (WAF) was prepared for the test. This was done by mixing the nominal load of 10 mg/L (real load = 10.3 mg/L) with the corresponding amount of dilution water and shaking vigorously for 24 hours. The resulting solution was filtrated through 0.45 μm PTFE filters. The lower treatments were prepared by dilution of this WAF with the corresponding amount of dilution water. This procedure is in agreement with the OECD guidance document no. 23 for the testing of mixtures which are toxic at low loading rates (≤ 1 mg/L). In a non-GLP pre-test at the loading rate 1 mg/L slight toxicity was observed.
The individual preparation of loading rates lower than 1 mg/L is technically not possible and therefore dilution of the WAF is the applicable method.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Strain/clone: STRAUS Berlin.
- Source: Umweltbundesamt Berlin. In-house breeding since 27. September 2007.
- Age: Females aged 0 – 24 hours.
- Feeding during test: no

ACCLIMATION
- Daphnia magna is bred in the LAUS GmbH throughout the year. The animals are kept for the use in toxicity tests. They multiply by parthenogenesis, thus being genetically identical.
- Acclimation conditions: Medium: M4-Medium (recipe of ELENDT). Photo period: 16/8 hours, using neon tubes. Temperature: 20 ± 2 °C. Medium renewal twice a week.
- Type and amount of food: Green algae (Desmodesmus subspicatus).


METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES:
- 19.25 hours before the start of the test, the adult animals were separated from the young. 1 hour before test start, the adults were caught with the help of a glass tube, and the new-born daphnia, aged between 0 and 18.25 hours, were sieved from the medium and immediately placed into a beaker containing dilution water. After the settling-in period, animals which showed no apparent damage were used for the test.
- Switching from M4-medium (husbandry) to Dilution water (test) has been shown not to cause any detrimental effects for test daphnia.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

250 mg CaCO3/L

Test temperature:

19.6 - 22.5 °C

pH:

7.6 - 8.1

Dissolved oxygen:

8.2 - 8.7 mg/L

Nominal and measured concentrations:

Nominal: 0.46, 1.0, 2.2, 4.6 and 10 mg/L
Calculated concentration from the geometric mean of the measured concentration of the highest tested concentration: 0.1, 0.3, 0.6, 1.2 and 2.6 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass beakers, nominal volume 50 mL, tall shape.
- Renewal rate of test solution (frequency/flow rate): None
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4 vessels, each containing 20 ± 3 mL test solution
- No. of vessels per control (replicates): 4 vessels, each containing 20 ± 3 mL dilution water

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Dilution water (Daphnia medium) with the following specification was used: CaCl2*2H2O 293.80 mg/L, MgSO4*7H2O 123.30 mg/L, NaHCO3 64.80 mg/L, KCl 5.80 mg/L. Resulting hardness in mmol/L: 2.502. Resulting hardness in mg CaCO3/L: 250. After preparation, the dilution water was aerated and the pH was measured. The pH was 7.8.
- Intervals of water quality measurement: The pH, the concentration of dissolved oxygen and the content of DOC in the test solutions were measured at the beginning and at the end of the test.

EFFECT PARAMETERS MEASURED
- After 24 and 48 hours, the immobilised daphnia were counted. Daphnia are considered immobilised when they do not perform any movements or are only able to move their antennae when the beaker is gently agitated. Daphnia which are trapped at the surface of the test solution are also considered immobilised.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

1.59 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

0.61 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

0.3 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

0.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

IMMOBILITY
- In the blank control, none of the daphnia were immobilised.

ANALYTICAL DETERMINATIONS
- Due to low solubility of test material only in the highest tested concentration (prepared WAF) test material was clearly determinable and was at 26 % of the nominal concentration at the beginning and at the end of the test. The lower concentrations (prepared by dilution of WAF) showed fluctuating DOC values which were similar to those of the blank control. Since toxicity was observed in the three highest concentrations, test material must have been present. Therefore, the determination of the biological results was based on the calculated concentrations from the geometric mean of the measured concentration of the highest tested concentration with the respective dilution factor. Geometric mean is calculated by multiplication of the n participating concentrations and taking the nth root.

VALIDITY
- Immobilisation in the controls may not exceed 10 %: Immobilisation in the controls was 0 %.
- The concentration of dissolved oxygen at the end of the test must be at least 3 mg/L: The lowest concentration of dissolved oxygen at the end of the test was 8.2 mg/L.
- The result of the test is considered valid.

Results with reference substance (positive control):

- Results with reference substance valid?: Yes
- ECx: The 24h-EC50 value was determined as 1.9 mg/L. The value was within the requested range for the 24h-EC50 between 0.6 - 2.1 mg/L of potassium dichromate K2Cr2O7 as specified in the OECD guideline.
- Since the fits showed sufficient statistical correspondence of the data with the dose-response-equation, this reference study can be considered valid.

Reported statistics and error estimates:

The estimation of the biological results was accomplished using the software ToxRat® Professional, version 3.2.1.

Table 1: Immobility

Nominal Concentration (mg/L)	Immobility 24 Hours					Immobility 48 Hours
	Absolute				%	Absolute				%
Blank control	0	0	0	0	0	0	0	0	0	0
0.46	0	0	0	0	0	0	0	0	0	0
1.0	0	0	0	0	0	0	0	0	0	0
2.2	1	0	0	0	5	1	1	5	2	45
4.6	1	1	0	0	10	5	5	5	5	100
10	4	5	5	5	95	5	5	5	5	100

 

Table 2: Measured Concentrations IC and TC

Nominal Concentration (mg/L)	Measured TC t = 0 h (mg/L)	Measured TC t = 48 h (mg/L)	Measured IC t = 0 h (mg/L)	Measured IC t = 48 h (mg/L)
Blank control	9.83	18.91	9.37	11.44
0.46	9.78	18.78	9.41	11.69
1.0	10.02	19.87	9.50	11.98
2.2	9.92	19.85	9.41	12.14
4.6	10.34	19.37	9.42	11.60
10	11.79	21.43	9.42	12.08

LOQ (Limit of quantification) TC = 5.43 mg/L

LOQ (Limit of quantification) IC = 2.03 mg/L

 

Table 3: Measured Concentrations DOC

Nominal Concentration (mg/L)	Measured DOC (TC-TI) t = 0 h (mg/L)	Measured DOC (TC-TI) t = 48 h (mg/L)	Measured DOC Minus Blank Control t = 0 h (mg/L)	Measured DOC Minus Blank Control t = 48 h (mg/L)
Blank control	0.47	7.47	-	-
0.46	0.38	7.09	-0.09	-0.38
1.0	0.52	7.89	0.05	0.42
2.2	0.51	7.71	0.05	0.24
4.6	0.92	7.77	0.45	0.30
10	2.37	9.35	1.90	1.88

 

Table 4: Calculated Test Material Concentrations

Nominal Concentration (mg/L)	Calculated Concentration Test Material t = 0 h (mg/L)	Calculated Concentration Test Material t = 48 h (mg/L)	% of Nominal Concentration t = 0 (%)	% of Nominal Concentration t = 48 (%)
Blank control	-	-	-	-
0.46	-0.1	-0.5	-28	-115
1.0	0.1	0.6	7	58
2.2	0.1	0.3	3	15
4.6	0.6	0.4	14	9
10	2.6	2.6	26	26

 

Table 5: Geometric Mean

Nominal Concentration (mg/L)	Geometric Mean of Measured Concentrations (mg/L)	Calculated Concentration from the Geometric Mean of the Measured Concentration of the Highest Tested Concentration
Blank control	-	-
0.46	n.d.	0.1
1.0	0.2	0.3
2.2	0.1	0.6
4.6	0.5	1.2
10	2.6	2.6

n.d. = not determinable due to mathematical reasons

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this study the EC50 value after 48 hours was 0.61 mg/L. The NOEC and LOEC were 0.3 and 0.6 mg/L, respectively.

Executive summary:

The toxicity of the test material to aquatic invertebrates was investigated in accordance with the standardised guidelines OECD 202 and EU Method C.2, under GLP conditions.

One valid experiment was performed using Daphnia magna STRAUS.

The study was performed using 5 concentrations of the test material ranging from 0.46 to 10 mg/L. For each test concentration and the blank control, 20 daphnia were exposed to the test material for 48 hours in a static test system. After 24 and 48 hours, the immobilised daphnia were counted.

Three concentrations showed toxicity between 45 and 100 % immobilisation. None of the animals were immobilised in the blank control.

Potassium dichromate K2Cr2O7 (CAS No. 7778-50-9) was used as positive control in a current reference study to assure that the test conditions are reliable.

Due to low solubility of test material only in the highest tested concentration (prepared WAF) test material was clearly determinable and was at 26 % of the nominal concentration at the beginning and at the end of the test. The lower concentrations (prepared by dilution of WAF) showed fluctuating DOC values which were similar to those of the blank control. Since toxicity was observed in the three highest concentrations, test material must have been present. Therefore, the determination of the biological results was based on the calculated concentrations from the geometric mean of the measured concentration of the highest tested concentration with the respective dilution factor.

Under the conditions of this study after 48 hours the EC50 value was 0.61 mg/L. The NOEC and LOEC were 0.3 and 0.6 mg/L, respectively.

Description of key information

Under the conditions of this study the EC50 value after 48 hours was 0.61 mg/L. The NOEC and LOEC were 0.3 and 0.6 mg/L, respectively.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.61 mg/L

Additional information

The toxicity of the test material to aquatic invertebrates was investigated in accordance with the standardised guidelines OECD 202 and EU Method C.2, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

One valid experiment was performed using Daphnia magna STRAUS.

The study was performed using 5 concentrations of the test material ranging from 0.46 to 10 mg/L. For each test concentration and the blank control, 20 daphnia were exposed to the test material for 48 hours in a static test system. After 24 and 48 hours, the immobilised daphnia were counted.

Three concentrations showed toxicity between 45 and 100 % immobilisation. None of the animals were immobilised in the blank control.

Potassium dichromate K2Cr2O7 (CAS No. 7778-50-9) was used as positive control in a current reference study to assure that the test conditions are reliable.

Due to low solubility of test material only in the highest tested concentration (prepared WAF) test material was clearly determinable and was at 26 % of the nominal concentration at the beginning and at the end of the test. The lower concentrations (prepared by dilution of WAF) showed fluctuating DOC values which were similar to those of the blank control. Since toxicity was observed in the three highest concentrations, test material must have been present. Therefore, the determination of the biological results was based on the calculated concentrations from the geometric mean of the measured concentration of the highest tested concentration with the respective dilution factor.

Under the conditions of this study after 48 hours the EC50 value was 0.61 mg/L. The NOEC and LOEC were 0.3 and 0.6 mg/L, respectively.