[2S-[2α,5α,6β(S*)]]-6-[[[[(4-ethyl-2,3-dioxopiperazin-1-yl)carbonyl]amino]phenylacetyl]amino]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16/07/2018 - 20/07/2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1075 (Freshwater and Saltwater Fish Acute Toxicity Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0 (control), 100 mg/L.
- Sampling method: The test concentration was analytically determined at the start and at the end of the renewal periods (0, 24, 48, 72, 96h) by HPLC-UV method.
- Sample storage conditions before analysis: No storage was necessary.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution with a concentration of 100 mg/L (nominal) was prepared with direct addition of the test item, mixed into the test medium (aquarium water) using ultrasonic bath (approximately 5 minutes). As a limit test was carried out, further dilution of stock solution was not performed.
- Eluate: aquarium water.
- Controls: The dilution water (circulated and filtered) was used without of addition of the test item.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): no.
- As the sample was only stable for 1 day, fresh test item solution was prepared by the method described above at each renewal period.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebrafish
- Strain: Brachydanio rerio
- Source: István Szent University, 2100 Gödöllő, Páter Károly u. 1, Hungary
- Age at study initiation (mean and range, SD): not specified.
- Length at study initiation (length definition, mean, range and SD): 2.5 – 2.8 cm
- Weight at study initiation (mean and range, SD): mean weight 0.22 – 0.26 g

ACCLIMATION
- Acclimation period: more than 12 days
- Acclimation conditions (same as test or not): same as test.
- Health during acclimation (any mortality observed): Fish were bred in a well-known fish farm, under disease- and parasite-controlled conditions. Fish were observed during the acclimatisation and test periods, the fish were healthy. There was no mortality of the population for seven days before the test.

FEEDING DURING TEST
- The fish were not fed during the test.

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Hardness:

194 mg/L (as CaCO3)

Test temperature:

20.5 – 22.3 ºC

pH:

7.81 – 8.34

Dissolved oxygen:

83 – 100 % of the air saturation value at the temperature used.

Nominal and measured concentrations:

- Nominal concentrations: 0 (control), 100 mg/L.
- Measured concentrations: 0 (control), 102 and 101 mg/L (at the start of the 48h-renewal periods); 91.7 and 96.2 mg/L (at the end of the renewal periods).

Details on test conditions:

TEST SYSTEM
- Test vessel: one aquarium for the test group and another one for the control group.
- Material, size, headspace, fill volume: fill volume 5L test solution.
- Renewal rate of test solution (frequency/flow rate): every 48h (as seen in study 17/340-316AN, the sample was only stable for 2 days in aquarium water).
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: The loading of the test aquaria was less than 1.0 g fish/L test solution at the start of the experiment.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: aquarium water (circulated and filtered)
- Culture medium different from test medium: no.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hours light and 8 hours darkness

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): The observations of mortality and sub-lethal effects of the fish were carried out in the following intervals: at 3, 6, 24, 48, 72 and 96 hours. The test conditions (pH, temperature, oxygen saturation) were measured daily (twice after 48 hours - at the end of the first as well as at the beginning of the second renewal period) during the test. The body weights of the fish were recorded before the introduction of fish. The body weight of 7 fish per aquarium was registered. The loading of the aquaria was calculated on the basis of these body weights (1.0 g fish/litre testing liquid).

TEST CONCENTRATIONS
- Range finding study: Four fish were exposed to each concentration of the test item plus a control, for 96 hours under semi-static conditions (with 24-hour renewal).
- Test concentrations: In the range-finding study, the concentrations tested were 0 (control), 0.1, 1, 10, 100 mg/L test item. Four fish were exposed to each concentration of the test item plus a control, for 96 hours under semi-static conditions (with 24-hour renewal, in absence of stability data).
- Results used to determine the conditions for the definitive study: Because no toxic response was observed during the preliminary range-finding test, only one test concentration (100 mg/L)) and one control group was tested in the definitive study in a semi-static system (limit test). As the measured concentrations deviated more than 20 per cent from the nominal at the end of the renewal periods, biological results are based on the measured test item concentration.

Reference substance (positive control):

not required

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 81.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LC100

Effect conc.:

> 81.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

81.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LOEC

Effect conc.:

> 81.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

24 h

Dose descriptor:

LC50

Effect conc.:

> 81.7 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

> 81.7 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

72 h

Dose descriptor:

LC50

Effect conc.:

> 81.7 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

- Behavioural abnormalities: No abnormal behaviour, appearance or any other effect on test animals were detected.
- Observations on body length and weight: There was no considerable difference observed concerning body weights between the groups. Body lengths were within the acceptable ranges.
- Mortality of control: 0/7.
- Other adverse effects control: no.
- Abnormal responses: 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.

Reported statistics and error estimates:

No statistical analysis was performed because of the lack of toxic effects. The LC50, NOEC, LOEC and LC100 were determined directly from the raw data.

Sublethal observations / clinical signs:

Table 3. Cumulative mortality data in the Limit Test.

Test Group	Cumulative Mortality (number of dead fish) [initial population = 7 fish]
	3h	6h	24h	48h	72h	96h
Control	0	0	0	0	0	0
81.7 mg/L (measured)	0	0	0	0	0	0

Table 4. Measured and calculated data of bodyweight.

Test Group	Measured weight of 7 fish (g)	Calculated mean weight of 1 fish (g)	Loading of testing aquarium (g fish/L testing liquid)
Control	1.53	0.22	0.31
81.7 mg/L (measured)	1.81	0.26	0.36

Table 5. Temperature.

Test Group	Measuring
	0h (1)	24h (2)	24 (1)	48 (2)	48 (1)	72 (2)	72 (1)	96 (2)
Control	21.4	20.5	21.3	20.8	21.5	20.8	21.6	20.5
81.7 mg/L (measured)	21.9	20.5	21.9	20.8	22.1	20.8	22.3	20.5

(1): measurement at the start of the renewal period in the fresh test medium. (2): measurement at the end of the renewal period in the old test medium.

Table 6. Dissolved oxygen concentration.

Test Group	Measuring
	0h (1)	24h (2)	24 (1)	48 (2)	48 (1)	72 (2)	72 (1)	96 (2)
Control	100	91	100	85	100	87	99	91
81.7 mg/L (measured)	98	89	100	83	100	82	98	88

(1): measurement at the start of the renewal period in the fresh test medium. (2): measurement at the end of the renewal period in the old test medium.

Table 7. pH.

Test Group	Measuring
	0h (1)	24h (2)	24 (1)	48 (2)	48 (1)	72 (2)	72 (1)	96 (2)
Control	8.34	8.11	8.30	8.04	8.11	8.04	8.23	7.96
81.7 mg/L (measured)	7.98	8.02	7.99	7.85	8.07	7.89	7.91	7.81

(1): measurement at the start of the renewal period in the fresh test medium. (2): measurement at the end of the renewal period in the old test medium.

Table 8. Body length of test fish at the end of the test.

Test Group	Body length [cm]
	1	2	3	4	5	6	7
Control	2.5	2.6	2.6	2.7	2.5	2.6	2.7
81.7 mg/L (measured)	2.6	2.8	2.7	2.5	2.7	2.7	2.6

Validity criteria fulfilled:

yes

Remarks:

All validity criteria were fulfilled; see 'Overall remarks'.

Conclusions:

The 96h-EC50 of the test item in zebrafish was found to be higher tan 81.7 mg/L and the NOEC was 81.7 mg/L (measured, geometric mean), both based on mortality.

Executive summary:

An acute toxicity test on zebrafish (Brachydanio rerio) was performed in accordance with OECD Guideline 203 / EU method C.1, under GLP conditions. Based on the results obtained in a preliminary range-finding test where no toxic response was observed, a Limit Test was carried out using only one test concentration (100 mg/L) and one control group in a semi-static system. The renewal period of the water was 48h, due to the stability of the test item. Seven fish per group were exposed to the test item or the control for 96 h. Test concentrations were analytically determined by means of a validated analytical method at the beginning and at the end of each renewal period and, as the measured concentrations deviated more than 20 per cent from the nominal at the end of the renewal periods, biological results are based on the measured test item concentration. All validity criteria were met. The 96h-EC50 of the test item in zebrafish was determined to be higher tan 81.7 mg/L and the NOEC was 81.7 mg/L (measured geometric mean), both based on mortality.

Description of key information

Key study. Method according to OECD 203 (limit test), GLP study. The 96h-EC50 of the test item in zebrafish was determined to be higher tan 81.7 mg/L and the NOEC was 81.7 mg/L (measured geometric mean), both based on mortality.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

81.7 mg/L

Additional information

Key study. An acute toxicity test on zebrafish (Brachydanio rerio) was performed in accordance with OECD Guideline 203 / EU method C.1, under GLP conditions. Based on the results obtained in a preliminary range-finding test where no toxic response was observed, a Limit Test was carried out using only one test concentration (100 mg/L) and one control group in a semi-static system. The renewal period of the water was 48h, due to the stability of the test item. Seven fish per group were exposed to the test item or the control for 96 h. Test concentrations were analytically determined (by means of a validated method) at the beginning and at the end of each renewal period and, as the measured concentrations deviated more than 20 per cent from the nominal at the end of the renewal periods, biological results are based on the measured test item concentration. All validity criteria were met. The 96h-EC50 of the test item in zebrafish was determined to be higher tan 81.7 mg/L and the NOEC was 81.7 mg/L (measured geometric mean), both based on mortality.