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Ecotoxicological information

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:
16 Nov 2018 - 19 Feb 2019
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:
other: Guidance document on aqueous-phase aquatic toxicity testing of difficult test chemicals, OECD series on testing and assessment number 23
Version / remarks:
2nd edition, adopted July 6, 2018
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: All test concentrations and control
- Sampling method: 2.0 mL from the approximate centre of the test vessels at t=0 h, t=24 h, and t=48 h.
- Sample storage conditions before analysis: Samples were stored in a freezer (≤-15°C) until analysis at the analytical laboratory of the Test Facility.
- Samples taken from the fresh solutions were taken from one replicate per test group. Upon renewal and at the end of the test, the 24-hour old solutions in the replicates were pooled per test group before sampling.
Vehicle:
yes
Remarks:
Acetonitrile
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method for Final Test: Water Accommodated Fractions (WAFs) were prepared by using stock solutions in acetonitrile (ACN) at nominal concentrations of 0.0032, 0.010, 0.032, 0.10, 0.32 and 1.0 mg/mL. Empty test vessels were individually spiked with 0.5 mL of the respective stock. Each replicate of the control received 0.5 mL blank ACN. The solvent was completely evaporated overnight prior to addition of the test medium to avoid modification of WAF-composition due to presence of a water-miscible solvent. Thereafter, 50 mL test medium was added to each vessel. Test solutions, including those of the control, were agitated for a period of two days to ensure maximum dissolution of test item in test medium. Vessels were sealed during agitation to prevent vaporization of test medium. All test solutions were generally clear and colourless at the end of the preparation procedure.
- Controls: Test medium without test item but with the additive used in the treatment of the stock solutions.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Acetonitrile
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): In some vessels small dark-coloured particles of undissolved material were observed on the bottom of the test solutions after preparation.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna
- Strain/clone: Straus 1820
- Source: In-house laboratory culture with a known history
- Method of breeding: cyclical parthenogenesis under specified breeding conditions
- Age of parental stock: > 2 weeks
- Age at test initiation: < 24 hours
- Validity of batch: Daphnids originated from a healthy stock, 2nd to 5th brood, showing no signs of stress such as mortality >20%, presence of males, ephippia or discoloured animals and there was no delay in the production of the first brood.

CULTIVATION
- Start of each batch: Approximately 250 newborn daphnids, i.e. less than 3 days old, were placed into 5 litres of M7 medium in an all-glass culture vessel.
- Maximum age of the cultures: 4 weeks
- Renewal of the cultures: After 7 days of cultivation, half of the medium twice a week.
- Temperature of medium: 18-22°C
- Feeding: Daily, a suspension of fresh water algae.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
180 mg/L (expressed as CaCO3)
Test temperature:
19 - 21°C
pH:
8.0 - 8.1
Dissolved oxygen:
8.8 - 10 mg/L
Nominal and measured concentrations:
Nominal loading rate: 0.032, 0.10, 0.32, 1.0, 3.2 and 10 mg/L
Average exposure concentrations: 0.010, 0.033, 0.14, 0.41, 1.7 and 5.4 mg/L. Upon renewal and at the end of the test, the measured concentrations in the 24-hour old solutions varied between 34 and 104% relative to the initial concentrations.Therefore, the average exposure concentration was calculated. See 'Any other information on materials and methods' for the calculation, and Table 1 in 'Any other information on results' for details on measured concentrations.
Details on test conditions:
TEST SYSTEM
- Test vessel: 100 mL, all-glass, containing 50 mL of test solution
- Aeration: no aeration
- Feeding: no feeding
- No. of organisms per vessel: 5 daphnids
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Introduction of daphnids: Within 1,5 hour after preparation of the test solutions.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Medium prepared with tap water purified by Reverse Osmosis and the following salts: CaCl2.2H2O: 211.5 mg/L, MgSO4.7H2O: 88.8 mg/L, NaHCO3: 46.7 mg/L, KCl: 4.2 mg/L
- Culture medium different from test medium: yes (M7)
- Intervals of water quality measurement: At the beginning and at the end of the test, for all concentrations and the control. In addition, in the freshly prepared and 24-hour old test solutions upon renewal. Temperature of medium: continuously in a temperature control vessel, beginning at the start of the test.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 h daily.

EFFECT PARAMETERS MEASURED
- Immobility (including mortality) at 24 h and at 48 h.

RANGE-FINDING TEST AND FULL TESTS
- Combined Limit/Range-Finding Test concentrations: untreated control and WSFs individually prepared at loading rates of 1.0, 10, 100 mg/L. No solvent was used in this test
- Based on the combined limit/range-finding test, a first and second full test were performed. Preparation of test item was similar to the combined limit/range-finding test. Daphnids were exposed to an untreated control and to WSFs individually prepared at loading rates of 0.46, 1.0, 2.2, 4.6, 10 and 22 mg/L. The first full test was not valid and the results of the second full test were not in agreement with what was expected based on the combined limit/range-finding test and preceding full test. The Final Test was performed using an alternative procedure for the preparation of the test solutions.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate, performed Jan 2019
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
0.14 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
mobility
Remarks on result:
other: 95% confidence interval: 0.11 - 0.19 mg/L
Duration:
48 h
Dose descriptor:
EL50
Effect conc.:
0.33 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% confidence interval: 0.26 - 0.43 mg/L
Details on results:
- Behavioural abnormalities: none observed in the control group.
- Mortality of control: none
- Analytical measurements: Samples taken from all test concentrations and the control were analysed by following a fragment ion (m/z 88.8) obtained from the most abundant ion trace (m/z 420.3). The concentrations measured in the freshly prepared solutions increased with increasing loading rate and were comparable at both occasions, indicating reproducibility of the preparation procedure. No samples were taken from WAFs prepared at 3.2 and 10 mg/L during the second 24-hour renewal interval, since all daphnids exposed to these loadings died during the first 24 hours of exposure. Upon renewal and at the end of the test, the measured concentrations in the 24-hour old solutions varied between 34 and 104% relative to the initial concentrations.
It should be noted that relevant reserve samples taken from WAFs prepared at loading rates of 1.0 mg/L and higher were analysed, since the measured concentrations in the regular samples fell below the LOQ due to inappropriate sample dilution. Analysed concentrations in the reserve samples were comparable to those in the regular samples, which were estimated by extrapolation of the calibration curve.
It should be noted that a small response was measured in the control samples throughout the test. This response is most likely caused by carry-over, since a similar response was detected in the blank Quality Control (QC) samples as well as in the analytical blanks. Therefore, it was assumed that no test item was present in the control solutions during the exposure.
Since the test item is a UVCB, the effect parameters were expressed based on loading rates. In addition, effect parameters were expressed in terms of measured concentrations, which correlated to the loading rate and recorded effects. Measurements were based on the most abundant signal at m/z 420.3, which corresponds with the epoxide distribution. By using the analysed concentrations for expression of EC50-values, it is assumed that this fraction is representative for the whole test item. However, it is unknown whether the analytically monitored constituent is responsible for the recorded effects. Therefore, these effect parameters should be interpreted with caution, since other test item constituents that are not analytically monitored might be responsible for the recorded effects as well.
- No immobility was observed in the control and at the two lowest test concentrations throughout the test, while complete immobility was found at the two highest test concentrations already after 24 hours of exposure. Microscopic observation revealed that all daphnids exposed to these test concentrations had died, and therefore, were not transferred to fresh solutions upon renewal. At the end of the test, 50% and 100% immobility was found at the WAFs prepared at loading rates of 0.32 and 1.0 mg/L, respectively. It should be noted that small dark-coloured particles of undissolved material were observed at the bottom of the test vessels of all test groups at the start of the test and upon renewal in the 24-hour old solutions. The origin of these particles is unknown and their presence did not correlate to the loading rate. In addition, these particles were observed in one of the control replicates as well, and thus, were assumed to be not test item related. Since no effects were observed in the control, it was considered that the particles had no contribution to the recorded effects.
See Table 2 in 'Any other information on results' for full details on immobility.
- All water quality parameters remained within the requirements as laid down in the study plan throughout the test.
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- The 48h-EC50 was 0.47 mg/L with a 95% confidence interval between 0.41 and 0.55 mg/L.
- The responses in this reference test with K2Cr2O7 are within the ranges of the expected responses at the different concentrations, i.e. the 48h-EC50 was between 0.28 and 0.75 mg/L. Hence, the sensitivity of this batch of D. magna was in agreement with ISO International Standard 6341, October 2012, and the historical data collected at the Test Facility.
Reported statistics and error estimates:
The 48h-EL50 was calculated from the weibits of the percentages of affected daphnids and the logarithms of the corresponding loading rates of the test item using the maximum likelihood estimation method. The 48h-EC50 was calculated similarly using average exposure concentrations instead.
ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used to perform the analysis.

Table 1: Measured Concentrations in Samples taken during the Final Test and Calculated Average Exposure Concentrations

 

Loading rate (mg/L)

Measured concentrations (mg/L)

Average exposure conc. (mg/L)

t=0h
(fresh)

t=24h
(old)

t=24h
(fresh)

t=48h
(old)

0.032

0.010

0.0059

0.013

0.011

0.010

0.10

0.058

0.020

0.050

0.021

0.033

0.32

0.17

0.12

0.16

0.12

0.14

1.0

0.42 (a)

0.43 (a)

0.45 (a)

0.34 (a)

0.41

3.2

1.8 (a)

1.6 (a)

2.0 (b)

n.d.

1.7

10

5.7 (a)

5.2 (a)

6.4 (b)

n.d.

5.4

(a) Analyzed concentration in the reserve sample.
(b) Estimated by extrapolation of the calibration curve. No reserve sample was analyzed, since no surviving daphnids were present after 24 hours of exposure.
n.d. – not determined, since no surviving daphnids were present after 24 hours of exposure.

Table 2: Number of Introduced Daphnids and Incidence of Immobility in the Final Test

 

Time (h)

Replicate


Loading rate (mg/L)

Control

0.032

0.10

0.32

1.0

3.2

10

0

A

5

5

5

5

5

5

5

B

5

5

5

5

5

5

5

C

5

5

5

5

5

5

5

D

5

5

5

5

5

5

5

Total introduced

20

20

20

20

20

20

20

 

24

A

0

0

0

0

0

5

5

B

0

0

0

0

1

5

5

C

0

0

0

0

2#

5

5

D

0

0

0

0

2

5

5

Total immobilised

0

0

0

0

5

20

20

Effect %

0

0

0

0

25

100

100

 

 

 

 

 

 

 

48

A

0

0

0

3#

5

n.d.

n.d.

B

0

0

0

3

5

n.d.

n.d.

C

0

0

0

2

5

n.d.

n.d.

D

0

0

0

2

5

n.d.

n.d.

Total immobilised

0

0

0

10

20

20

20

Effect %

0

0

0

50

100

100

100

#Microscopic observation revealed no test item attached to the daphnids.
n.d – not determined, daphnids were removed at 24 hours after being scored as dead using a microscope.

Validity criteria fulfilled:
yes
Remarks:
See 'Overall remarks' for details on validity criteria.
Conclusions:
The 48h-EL50 for Daphnia magna exposed to the test substance was 0.33 mg/L (95% confidence interval between 0.26 and 0.43 mg/L).

It should be noted that the CMC (used here as water solubility) reported is 310 mg/L and that it was decided to follow the WAF approach because the test item is a UVCB and was observed to be not completely soluble in test medium.
These observations in relation to the solubility appear to be contradictory and therefore as worst-case the effect data based on geometric mean measured values will be used in stead. The highest nominal WAF concentration of 10 mg/L is about a factor 31 below the measured water solubility and it is therefore assumed that the test substance was actually completely dissolved and that the measured concentration for the most abundant ion trace (m/z 420.3) is representative for the availability of the whole test item.
The 48h-EC50 based geometric mean measured concentrations is 0.14 (0.11 - 0.19) mg/L. This value will be used as key value for chemical safety assessment. 
Executive summary:

The objective of the study was to evaluate BEROL 1872 for its ability to generate acute toxic effects on the mobility of Daphnia magna during an exposure period of 48 hours and, if possible, to determine the EL50 at 24 and 48 hours of exposure.

The study procedures described in this report were based on the OECD guideline No. 202, 2004. In addition, procedures were based on the test methods described in the OECD series on testing and assessment number 23, 2018.

The batch of BEROL 1872 tested was a light-yellow liquid UVCB and was not completely soluble in test medium at the loading rates initially prepared. Water Accommodated Fractions (WAFs) were individually prepared at loading rates in the range of 0.032 to 10 mg/L and used as test concentrations. Test solutions were prepared by using stock solutions in acetonitrile (ACN). Since the test item is a UVCB, the solvent was completely evaporated prior to addition of the test medium to avoid modification of WAF-composition due to presence of a water-miscible solvent.

A final test was performed based on the results of a preceding combined limit/range-finding test and two full tests. Twenty daphnids per group (5 per replicate, quadruplicate) were exposed to a solvent control and to WAFs prepared at loading rates of 0.032, 0.10, 0.32, 1.0, 3.2 and 10 mg/L. The total exposure period was 48 hours and samples for analytical confirmation of exposure concentrations were taken at the start and at the end of the test.

No immobility was observed in the control and at the two lowest test concentrations throughout the test, while complete immobility was found at the two highest test concentrations already after 24 hours of exposure. At the end of the test, 50% and 100% immobility was found at the WAFs prepared at a loading rates of 0.32 and 1.0 mg/L, respectively.

Samples taken from all test concentrations and the control were analysed by following a fragment ion (m/z 88.8) obtained from the most abundant ion trace (m/z 420.3). The concentrations measured in the freshly prepared solutions increased with increasing loading rate and were comparable at both occasions, indicating reproducibility of the preparation procedure. Since the test item is a UVCB, effect parameters were expressed in terms of loading rates and average exposure concentrations, which are indicative for the analytically monitored fraction of the test item.

The study met the acceptability criteria prescribed by the study plan and was considered valid.

In conclusion, the 48h-EL50 for Daphnia magna exposed to BEROL 1872 was 0.33 mg/L (95% confidence interval between 0.26 and 0.43 mg/L).

It should be noted that the CMC (used here as water solubility) reported is 310 mg/L and that it was decided to follow the WAF approach because the test item is a UVCB and was observed to be not completely soluble in test medium.

These observations in relation to the solubility appear to be contradictory and therefore as worst-case the effect data based on geometric mean measured values will be used in stead. The highest nominal WAF concentration of 10 mg/L is about a factor 31 below the measured water solubility and it is therefore assumed that the test substance was actually completely dissolved and that the measured concentration for the most abundant ion trace (m/z 420.3) is representative for the availability of the whole test item.

The 48h-EC50 based geometric mean measured concentrations is 0.14 (0.11 - 0.19) mg/L. This value will be used as key value for chemical safety assessment.

Description of key information

The 48h-EL50 for Daphnia magna exposed to the test substance was 0.33 mg/L (95% confidence interval between 0.26 and 0.43 mg/L).

It should be noted that the CMC (used here as water solubility) reported is 310 mg/L and that it was decided to follow the WAF approach because the test item is a UVCB and was observed to be not completely soluble in test medium.

These observations in relation to the solubility appear to be contradictory and therefore as worst-case the effect data based on geometric mean measured values will be used in stead. The highest nominal WAF concentration of 10 mg/L is about a factor 31 below the measured water solubility and it is therefore assumed that the test substance was actually completely dissolved and that the measured concentration for the most abundant ion trace (m/z 420.3) is representative for the availability of the whole test item.

The 48h-EC50 based geometric mean measured concentrations is 0.14 (0.11 - 0.19) mg/L. This value will be used as key value for chemical safety assessment.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
0.14 mg/L

Additional information

The objective of the study was to evaluate BEROL 1872 for its ability to generate acute toxic effects on the mobility of Daphnia magna during an exposure period of 48 hours and, if possible, to determine the EL50 at 24 and 48 hours of exposure.

The study procedures described in this report were based on the OECD guideline No. 202, 2004. In addition, procedures were based on the test methods described in the OECD series on testing and assessment number 23, 2018.

The batch of BEROL 1872 tested was a light-yellow liquid UVCB and was not completely soluble in test medium at the loading rates initially prepared. Water Accommodated Fractions (WAFs) were individually prepared at loading rates in the range of 0.032 to 10 mg/L and used as test concentrations. Test solutions were prepared by using stock solutions in acetonitrile (ACN). Since the test item is a UVCB, the solvent was completely evaporated prior to addition of the test medium to avoid modification of WAF-composition due to presence of a water-miscible solvent.

A final test was performed based on the results of a preceding combined limit/range-finding test and two full tests. Twenty daphnids per group (5 per replicate, quadruplicate) were exposed to a solvent control and to WAFs prepared at loading rates of 0.032, 0.10, 0.32, 1.0, 3.2 and 10 mg/L. The total exposure period was 48 hours and samples for analytical confirmation of exposure concentrations were taken at the start and at the end of the test.

No immobility was observed in the control and at the two lowest test concentrations throughout the test, while complete immobility was found at the two highest test concentrations already after 24 hours of exposure. At the end of the test, 50% and 100% immobility was found at the WAFs prepared at a loading rates of 0.32 and 1.0 mg/L, respectively.

Samples taken from all test concentrations and the control were analysed by following a fragment ion (m/z 88.8) obtained from the most abundant ion trace (m/z 420.3). The concentrations measured in the freshly prepared solutions increased with increasing loading rate and were comparable at both occasions, indicating reproducibility of the preparation procedure. Since the test item is a UVCB, effect parameters were expressed in terms of loading rates and average exposure concentrations, which are indicative for the analytically monitored fraction of the test item.

The study met the acceptability criteria prescribed by the study plan and was considered valid.

In conclusion, the 48h-EL50 for Daphnia magna exposed to BEROL 1872 was 0.33 mg/L (95% confidence interval between 0.26 and 0.43 mg/L).

It should be noted that the CMC (used here as water solubility) reported is 310 mg/L and that it was decided to follow the WAF approach because the test item is a UVCB and was observed to be not completely soluble in test medium.

These observations in relation to the solubility appear to be contradictory and therefore as worst-case the effect data based on geometric mean measured values will be used in stead. The highest nominal WAF concentration of 10 mg/L is about a factor 31 below the measured water solubility and it is therefore assumed that the test substance was actually completely dissolved and that the measured concentration for the most abundant ion trace (m/z 420.3) is representative for the availability of the whole test item.

The 48h-EC50 based geometric mean measured concentrations is 0.14 (0.11 - 0.19) mg/L. This value will be used as key value for chemical safety assessment.