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Short-term toxicity to fish

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Reference
Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 Jan 2019 - 19 Feb 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Version / remarks:
1992
Deviations:
no
Qualifier:
according to
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
Analytical monitoring:
yes
Details on sampling:
- Concentrations: All test concentrations and the control; stock solutions in acetonitrile used for preparation of fresh solutions for commencement of the test and third renewal after 72 hours of exposure were sampled as well.
- Sampling method: 2.0 mL from the approximate centre of the test vessels, taken at the start of the test and after 72 hours from the freshly prepared solutions, and at the first renewal (t=24 h) and the end of the test from the 24-h old solutions.
- Sample storage conditions before analysis: Samples were stored in a freezer (≤-15°C) until analysis at the analytical laboratory of the Test Facility.
Vehicle:
yes
Remarks:
Acetonitrile
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Water Accommodated Fractions (WAFs) were prepared by using stock solutions in acetonitrile (ACN) at nominal concentrations of 0.11, 0.23, 0.50, 1.1 and 2.3 mg/mL. Empty test vessels (i.e. 10 L all-glass fish tanks) were individually spiked with 10 mL of the respective stock. The tank of the control group received 10 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, 5 litre test medium was added to each tank. Test solutions, including those of the control, were magnetically stirred for a period of two days to ensure maximum dissolution of test item in test medium. Tanks were sealed during stirring using a plastic plate and silicon lubricant to prevent vaporization of test medium. All test solutions were clear and colourless at the end of the preparation procedure.
Upon renewal of test solutions after 24 hours of exposure, undissolved material was observed at the surface of the fresh solutions prepared at loading rates of 1.0 mg/L and higher. Similar material was observed at loading rates of 2.2 and 4.6 mg/L upon renewal after 48 hours and at loading rates of 0.46 mg/L and higher upon renewal after 72 hours. In addition, this material was also seen in the freshly prepared control solution upon the third renewal after 72 hours of exposure. No undissolved material was however observed in the solutions prepared for commencement of the test. Consequently, no separation step was applied in order not to change the preparation procedure for test solutions between renewals.
- Controls: Test medium without test item but with addition of the solvent used in the treatment of the stock solutions (solvent control).
Test organisms (species):
Cyprinus carpio
Details on test organisms:
TEST ORGANISM
- Common name: carp (Cyprinus carpio Linnaeus 1758)
- Source: "De Haar Vissen", Zodiac, proefacc. Wageningen University and Research Centre, The Netherlands
- Length at study initiation: 2.9 +/- 0.1 cm (Final test)
- Weight at study initiation: 0.27 +/- 0.07 g (Final test)
- Method of breeding: F1 from a single parent-pair bred in UV-treated water
- Total fish used: 51

HOLDING
- Quarantine/Acclimatisation: At least 12 days after delivery
- Medium: Adjusted ISO medium, formulated using tap-water purified by reverse osmosis
- Feeding: Daily with pelleted fish food (Essence 300-500 µm)
- Validity of batch: In the batch of fish used for the test, mortality during the seven days prior to the start of the test was less than 5%.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Hardness:
180 mg/L (expressed as CaCO3)
Test temperature:
21 - 22 °C
pH:
Fresh solutions: 8.0 - 8.1
Spent solutions: 7.6 - 8.2
Dissolved oxygen:
Fresh solutions: 8.2 - 8.8 mg/L
Spent solutions: 5.4 - 7.9 mg/L
Nominal and measured concentrations:
Nominal loading rate: 0.22, 0.46, 1.0, 2.2, 4.6 mg/L.
See Table 1 in 'Any other information on results', and text in 'Details on results' for details on measured concentrations.
Details on test conditions:
TEST SYSTEM
- Test vessel: 10 litres, all-glass, containing 5 litres of test solution
- Test type: semi-static, with daily renewal of test solutions
- Aeration: no aeration
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: 0.38 g fish/L, i.e. 7 fish per 5 litres of test medium

TEST MEDIUM / WATER PARAMETERS
- Test medium: Adjusted ISO medium
- Culture medium different from test medium: no
- Intervals of water quality measurement: Dissolved oxygen content, pH and temperature were measured daily in vessels with surviving fish, beginning at the start of the test (day 0).

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 h daily
- Feeding: No feeding from 24 hours prior to the test and during the total test period
- Introduction of fish: Within 1¼ hour after preparation of the test media from a holding tank with comparable water quality parameters and pH and temperature differences between test and holding tank media of less than 1.0 unit and 1.0°C. Upon each renewal of test solutions, fish were transferred to the fresh solutions within one hour after preparation.

TERMINAL PROCEDURES
- Euthanasia: At the end of the test, the surviving fish were rapidly killed by exposing them to ca. 1.2% ethylene glycol monophenylether in water.

EFFECT PARAMETERS MEASURED
- Mortality and other effects: At 2, 24, 48, 72 and 96 hours following the start of exposure. In addition, every afternoon from day 0 to observe for any dead or severely distressed fish. Dead fish were removed when observed.

TEST CONCENTRATIONS
- Range finding study test concentrations: WSFs individually prepared at loading rates of 1.0, 10 and 100 mg/L. The preparation of test solutions differed from the method used in the Final Test.
- Results used to determine the conditions for the definitive study: yes
Reference substance (positive control):
yes
Remarks:
Pentachlorophenol (performed Mar 2018)
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
>= 0.04 - <= 0.09 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
mortality
Remarks on result:
other: Estimated range for LC50 based on average exposure concentrations (measured)
Duration:
96 h
Dose descriptor:
LL50
Effect conc.:
3.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: 95% confidence interval: 2.2 - 4.6 mg/L
Details on results:
- Measured concentrations: 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).
Samples taken from ACN stock solutions prepared at the start of the test showed that measured concentrations varied between 69 and 79% of nominal concentrations, while concentrations in the stocks prepared for the third renewal period were in the range of 75 - 92% relative to nominal concentrations. Hence, stocks used for spiking of test concentrations were at or just below the required concentrations.
Measured concentrations in freshly prepared solutions (t=0 and t =72 h) ranged between 0.22 and 0.61 mg/L for the loading rates of 1.0 mg/L and higher. Loading rates prepared at 0.22 and 0.46 mg/L showed very low measured concentrations (<10 µg/L), except for 0.46 mg/L at t=72 h with a measured concentration of 0.18 mg/L. Measured concentrations in 24-hour old samples all decreased showing a very high fluctuation in the actual decrease. It was unclear what the cause for this fluctuation was. A plausible reason might be that solutions were not fully homogeneous when taking samples, despite the fact that the correct amount of introduced test item was mixed with test medium whilst preparing the test concentrations. This might also be the reason why the sample taken on day 2 (t=54 h) of the test from the highest test concentration showed an unexpected high concentration of 2.7 mg/L (mean of duplicate samples).
- Since the test item is a UVCB, effect parameters are to be expressed based on loading rates. Samples were analysed by following a fragment ion (m/z 88.8) obtained from the most abundant ion trace (m/z 420.3). Any measured concentrations should be considered only as indicative and cannot be reliably used other than for showing that loading rates as prepared contained increasing concentrations of the test item. The 96h-LC50 was expected between 0.04 and 0.09 mg/L, i.e. between average exposure concentrations (geometric mean) determined for the WAFs prepared at 2.2 and 4.6 mg/L during the first renewal interval. Lowest concentrations were measured during this period, and therefore, the estimated range for the 96h-LC50 can be considered as a worst-case approximation. As it is unknown which fraction of the test item is responsible for the observed mortality, effect parameters based on measured concentrations should be interpreted with caution.
- No mortality was observed in the control and at the four lowest test concentrations throughout the exposure period. Five fish exposed to a loading rate of 4.6 mg/L were found dead after 48 hours of exposure. The two surviving fish at this concentration were immobilized after 48 hours. Complete mortality was observed at this concentration after 72 hours exposure.
In addition, two fish exposed to a loading rate of 2.2 mg/L appeared to have lost their equilibrium as they were swimming sideways and upside down after 72 and 96 hours of exposure. These fish clearly showed adverse effects compared to the fish in the control group, however, survived the entire exposure period. See Table 2 in 'Any other information on results' for details mortality and Table 3 for details on clinical effects.
- It should be noted that the floating layer of undissolved material seen after preparation of test solutions was also observed in the 24-hour old WAFs prepared at loading rates of 2.2 and 4.6 mg/L after 48 and 72 hours of exposure. Nevertheless, this material was floating on the surface of the test solutions and was therefore not in direct contact with the fish. Since such layer was also found at a test concentration at which no mortality was found, it was considered that fish were not directly exposed to the undissolved material floating at the surface of the solutions.
- 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
- Mortality: 0% at 0.10 mg/L PCP; 100% at 0.22 mg/L PCP; 100% at 0.46 mg/L PCP
- LC50: 0.15 mg/L, 95% confidence interval 0.10 - 0.22 mg/L
- The range of the 96h-LC50 for carp is generally between 0.10 mg/L and 0.46 mg/L based on historical data of reference tests performed by the Test Facility. Hence, the sensitivity of carp originating from the present batch for PCP falls within the range of sensitivities generally observed during the past years.
Reported statistics and error estimates:
- No 24h-LL50 could be determined since mortality was below 50% after 24 hours of exposure (LL50 > maximum loading tested).
- The 48h-LL50 could not be determined using a regression method. Instead, the 48h-LL50 was calculated by applying the Spearman-Karber procedure (non-linear; without trimming) on the mortality percentages and the logarithms of the corresponding loading rates of the test item. ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used to perform the analysis.
- The 72h and 96h-LL50 could not be determined using statistical analysis. This was because there was no concentration between the highest concentration (A) at which 0% mortality and the lowest concentration (B) at which 100% mortality occurred after 72 and 96 hours of exposure. Instead, the LL50 was calculated as (AB)½, with A and B being limits of the 95% confidence interval.

Table 1: Measured Concentrations in Samples taken during the Final Test

Loading rate (mg/L)

Measured concentration (mg/L)

t=0h
 (fresh)

t=24h
(old)

t=54h
(old) (a)

t=72h (fresh)

t=96
(old)

0.22

<LOD (d)

0.0015 (e)

 

0.0011 (e)

<LOD (d)

0.46

<LOD (d)

<LOD (d)

 

0.18 (b)

0.0085 (e)

1.0

0.22

0.038 (b)

 

0.33

0.084

2.2

0.35

0.0039 (b,e)

 

0.61

0.47

4.6

0.52

0.017 (b)

2.7 (b,c)

n.d.

n.d.

(a) Sample was taken after all fish were found dead from the 6-hour old solution.
(b) Mean of analyzed concentrations in the regular and reserve sample.
(c) Analysis of the reserve sample revealed a concentration of 2.4 mg/L, which is comparable to the initial analyzed concentration of 3.1 mg/L.
(d)
Limit of Detection (LOD) was 0.00079 mg/L on the day of sample analysis.
(e) Estimated by extrapolation of the calibration curve.

n.d. – not determined, since no surviving fish were present at this concentration.

Table 2: Incidence of Mortality and Total Mortality during the Final Test


Loading rate (mg/L)

Initial
number
of fish

Cumulative mortality

Total
Mortality (%)

2h

24h

48h

72h

96h

Control

7

0

0

0

0

0

0

0.22

7

0

0

0

0

0

0

0.46

7

0

0

0

0

0

0

1.0

7

0

0

0

0

0

0

2.2

7

0

0

0*

0*

0

0

4.6

7

0

0

5*

7

7

100

* A floating layer of undissolved material was observed at the surface of the 24-hour old solutions upon renewal.

Table 3: Clinical Effects Observed in the Final Test


Loading rate (mg/L)

Time of recording (hours)

Specification of effects

Relative
number

2.2

72 – 96

Loss of equilibrium

2

4.6

48

Immobile

2

Validity criteria fulfilled:
yes
Remarks:
See 'Overall remarks' for details on validity criteria.
Conclusions:
The 96h-LL50 for carp (Cyprinus carpio) exposed to BEROL 1872 applying the WAF approach was 3.2 mg/L with a 95%-confidence interval ranging from 2.2 to 4.6 mg/L, which was already reached after 72 hours of exposure.

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 appear to be contradictory and therefore as worst-case the geometric mean measured values will be used in stead. The highest nominal WAF concentration of 4.6 mg/L is about a factor 67 below the 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 96h-LC50 based geometric mean measured concentrations lies between 0.04 and 0.09 mg/L i.e. between average exposure concentrations (geometric mean) determined for the WAFs prepared at 2.2 and 4.6 mg/L during the first renewal interval. Lowest concentrations were measured during this period, and therefore, the estimated range for the 96h-LC50 can be considered as a worst-case approximation.
Executive summary:

The objective of the study was to evaluate BEROL 1872 for its ability to generate acute toxic effects in Cyprinus carpio during an exposure period of 96 hours and, if possible, to determine the LL50 at all observation times.

The study procedure described in this report was based on the OECD guideline No. 203, 1992. 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 0.22 to 4.6 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 range-finding test. Seven fish per group (single tank, non-replicated) were exposed to a solvent control and to WAFs prepared at loading rates of 0.22, 0.46, 1.0, 2.2 and 4.6 mg/L. The total exposure period was 96 hours. Samples for analytical confirmation of actual exposure concentrations were taken during two renewal intervals from freshly prepared and 24-hour old solutions.

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 measured concentrations in the freshly prepared solutions increased with increasing loading rate in the freshly prepared solutions, which indicates proper preparation of test solutions. In addition, the analysed fraction of the test item correlated to the recorded mortality of fish. Since the test item is a UVCB, the effect parameters were expressed in terms of loading rates. For risk assessment purposes, an estimated range for the 96h-LC50 based on average exposure concentrations was provided.

No mortality was observed in the control and at the four lowest test concentrations throughout the exposure period. Five fish exposed to a loading rate of 4.6 mg/L were found dead after 48 hours of exposure. The two surviving fish at this concentration were immobilized after 48 hours of exposure. Complete mortality was observed at this concentration after 72 hours exposure. The study met the acceptability criteria prescribed by the study plan and was considered valid.

In conclusion, the 96h-LL50 for carp (Cyprinus carpio) exposed to BEROL 1872 was 3.2 mg/L with a 95%-confidence interval ranging from 2.2 to 4.6 mg/L, which was already reached after 72 hours of exposure.

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 4.6 mg/L is about a factor 67 below the 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 96h-LC50 based geometric mean measured concentrations lies between 0.04 and 0.09 mg/L i.e. between average exposure concentrations (geometric mean) determined for the WAFs prepared at 2.2 and 4.6 mg/L during the first renewal interval. Lowest concentrations were measured during this period, and therefore, the estimated range for the 96h-LC50 can be considered as a worst-case approximation.

Description of key information

The 96h-LL50 for carp (Cyprinus carpio) was 3.2 mg/L, with a 95%-confidence interval ranging from 2.2 to 4.6 mg/L, which was already reached after 72 hours of exposure.

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 4.6 mg/L is about a factor 67 below the 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 96h-LC50 based geometric mean measured concentrations lies between 0.04 and 0.09 mg/L i.e. between average exposure concentrations (geometric mean) determined for the WAFs prepared at 2.2 and 4.6 mg/L during the first renewal interval. Lowest concentrations were measured during this period, and therefore, the estimated range for the 96h-LC50 can be considered as a worst-case approximation. The mean value of 0.065 mg/L will be used as key value for chemical safety assessment.

Key value for chemical safety assessment

LC50 for freshwater fish:
0.065 mg/L

Additional information

The objective of the study was to evaluate BEROL 1872 for its ability to generate acute toxic effects in Cyprinus carpio during an exposure period of 96 hours and, if possible, to determine the LL50 at all observation times.

The study procedure described in this report was based on the OECD guideline No. 203, 1992. 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 0.22 to 4.6 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 range-finding test. Seven fish per group (single tank, non-replicated) were exposed to a solvent control and to WAFs prepared at loading rates of 0.22, 0.46, 1.0, 2.2 and 4.6 mg/L. The total exposure period was 96 hours. Samples for analytical confirmation of actual exposure concentrations were taken during two renewal intervals from freshly prepared and 24-hour old solutions.

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 measured concentrations in the freshly prepared solutions increased with increasing loading rate in the freshly prepared solutions, which indicates proper preparation of test solutions. In addition, the analysed fraction of the test item correlated to the recorded mortality of fish. Since the test item is a UVCB, the effect parameters were expressed in terms of loading rates. For risk assessment purposes, an estimated range for the 96h-LC50 based on average exposure concentrations was provided.

No mortality was observed in the control and at the four lowest test concentrations throughout the exposure period. Five fish exposed to a loading rate of 4.6 mg/L were found dead after 48 hours of exposure. The two surviving fish at this concentration were immobilized after 48 hours of exposure. Complete mortality was observed at this concentration after 72 hours exposure. The study met the acceptability criteria prescribed by the study plan and was considered valid.

In conclusion, the 96h-LL50 for carp (Cyprinus carpio) exposed to BEROL 1872 was 3.2 mg/L with a 95%-confidence interval ranging from 2.2 to 4.6 mg/L, which was already reached after 72 hours of exposure.

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 4.6 mg/L is about a factor 10 below the 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 96h-LC50 based geometric mean measured concentrations lies between 0.04 and 0.09 mg/L i.e. between average exposure concentrations (geometric mean) determined for the WAFs prepared at 2.2 and 4.6 mg/L during the first renewal interval. Lowest concentrations were measured during this period, and therefore, the estimated range for the 96h-LC50 can be considered as a worst-case approximation. The mean value of 0.065 mg/L will be used as key value for chemical safety assessment.