N-bromosuccinimide

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 Oct 2017 - 03 Feb 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)

Version / remarks:

1992

Deviations:

no

Remarks:

The mean length of fish used for the final test was 3.6 ± 0.5 cm, which exceeds the range given in the guideline by 0.1 cm (i.e. 3.0 ± 1.0 cm). This deviation is considered negligible and had no effect on the outcome or validity of the study.

Qualifier:

according to guideline

Guideline:

other: Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment number 23

Version / remarks:

2000

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 5.0 mg/L stock solutions only.
- Sampling method: 0.4 mL from the approximate centre of the vessel.
- Frequency: At the start of the test and after 72 hours from the freshly prepared stock solutions, and at the first renewal (t=24h) and the end of the test from the 24-hour old stock solutions.
- Sample storage conditions before analysis: Not applicable, samples were transferred to the analytical laboratory at the Test Facility and analysed on the day of sampling.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Preparation of test solutions started with the highest concentration of 5.0 mg/L (Final Test). A 28-43 minute period of magnetic stirring was applied to accelerate dissolution of the test item in medium. Lower test concentrations were prepared by subsequent dilutions of the highest concentration in test medium. All test solutions were clear and colorless at the end of the preparation procedure.
The preparation procedure was performed under dimmed light and glassware was covered with aluminum foil.
- Controls: Test medium without test item or other additives (blank-control)
-

Test organisms (species):

Cyprinus carpio

Details on test organisms:

TEST ORGANISM
- Common name: Carp
- Strain: Cyprinus carpio Linnaeus 1758
- Source: Zodiac, proefacc, "De Haar Vissen", Wageningen University and Research Centre, The Netherlands
- Length at study initiation: 3.6 ± 0.5 cm
- Weight at study initiation: 0.60 ± 0.22 g
- Method of breeding: F1 from a single parent-pair bred in UV-treted water.

HOLDING
- Acclimation period: At least 12 days after delivery
- Medium: Adjusted ISO medium, formulated using RO-water (tap-water purified by reverse osmosis)
- Feeding: Daily with pelleted fish food (Cyprico Crumble Excellent (300-500 um)
- Water quality parameters: Within the optimum limits for the respective fish species.
- Health during acclimation (any mortality observed): 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 CaCO3/L

Test temperature:

21 - 23°C

pH:

7.5 - 8.0

Dissolved oxygen:

Fresh medium (day 0,1,2,3): 8.7 - 9.5 mg/L
Old medium (day 1,2,3,4): 7.2 - 7.9 mg/L

Nominal and measured concentrations:

Nominal concentrations: 0.10, 0.22, 0.46, 1.0, 2.2 mg/L
Average exposure concentrations: 0.058, 0.13, 0.27, 0.58, 1.3 mg/L (calculated using a a correction factor based on geometric mean measured concentration in the stock solution (5.00 mg/L) compared to nominal concentration). See Table 1 in 'Any other information on results' for measured concentrations. Effect parameters were calculated based on both the nominal concentrations and the average exposure concentrations.

Details on test conditions:

TEST SYSTEM
- Test vessel: 10 litres, all-glass, containing 9.5 litres of test solution; air-tight closed.
- Aeration: no aeration
- Feeding: No feeding from 24 hours prior to the test and during the total test period.
- Renewal of test solution: Daily
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading: 0.44 g fish/L
- Introduction of fish: Within 16 minutes 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 0.5 unit and 1.0°C.
- Terminal procedure: At the end of the test the surviving fish were rapidly killed by exposing them to ca. 1.2% ethylene glycol monophenylether in water.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Adjusted ISO medium
- Culture medium different from test medium: no
- Intervals of water quality measurement: Dissolved oxygen, pH and temperature: At the start of the test and upon renewal in fresh and old solutions in all vessels with surviving fish, beginning at the start of the test (day 0).

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hours dimmed light per day

EFFECT PARAMETERS MEASURED
- Mortality and other effects: at 3¾, 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
- Spacing factor for test concentrations: 2.2
- Range finding study test concentrations: 1.0, 10, 100 mg/L (first range-finding test); 0.0050, 0.050, 0.50 mg/L (second range-finding test)
- Results used to determine the conditions for the definitive study: yes.

Reference substance (positive control):

yes

Remarks:

Pentachlorophenol (PCP, performed March 2018)

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

0.65 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95% confidence interval: 0.48-0.99 mg/L

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

0.4 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95% confidence interval: 0.27-0.58 mg/L

Details on results:

- No mortality or other effects were observed in the control and the three lowest concentrations tested. All fish exposed to the highest concentration were found dead after the first 3¾ hours of exposure. Fish exposed to the nominal concentration of 1.0 mg/L did not survive the first 24 hours of exposure.
- Clinical effects observed: see Table 3 in 'Any other information on results'.
- All water quality parameters remained within the limits as prescribed by the Study Plan.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- 96h-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 and 0.46 mg/L based on historical data of reference tests performed approximately every 3 months from April 1988 until the end of 2000, and annually since then. 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:

LC50 value (based on nominal concentrations): Probit analysis, maximum likelihood estimation method.
3¾-LC50 value (based on average exposure concentrations): Spearman-Karber procedure (without trim).
24-96h-LC50 values based on average exposure concentrations could not be determined using the maximum likelihood estimation method or the Spearman-Karber procedure. 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. Instead, the LC50 was calculated as (AB)½, with A and B being limits of the 95% confidence interval.
ToxRat Professional v 3.2 (ToxRat Solutions® GmbH, Germany) was used to perform the analyses.

Sublethal observations / clinical signs:

Table 1: Full Test: Test Samples

Time of sampling [days]	Date of sampling	Date of analysis	Concentration [mg/L]		Relative to nominal [%]	Relative to initial [%]
			Nominal	Analyzed
0 (fresh)	29 Jan 2018	29 Jan 2018	5.00	4.24	85
1 (old)	30 Jan 2018	30 Jan 2018	5.00	1.8 #	36	43
3 (fresh)	01 Feb 2018	01 Feb 2018	5.00	4.92	98
4 (old)	02 Feb 2018	02 Feb 2018	5.00	1.8 #	37	37

# Estimated value, calculated by extrapolation of the calibration curve.

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

Nominal concentration N-BROMOSUCCINIMIDE (mg/L)	Initial number of fish	Cumulativemortality					Total Mortality (%)
		3¾h	24h	48h	72h	96h
Control	7	0	0	0	0	0	0
0.10 (0.058)	7	0	0	0	0	0	0
0.22 (0.13)	7	0	0	0	0	0	0
0.46 (0.27)	7	0	0	0	0	0	0
1.0 (0.58)	7	2	7	7	7	7	100
2.2 (1.3)	7	7	7	7	7	7	100

( ) – average exposure concentration (mg/L)

 

Table 3: Clinical Effects Observed During the Final Test

Nominal concentration N-BROMOSUCCINIMIDE (mg/L)	Time of recording (hours)	Specification of effects	Relative number
1.0 (0.58)	3¾	Loss of equilibrium Immobile	2/5 1/5

() – average exposure concentration (mg/L)

Validity criteria fulfilled:

yes

Conclusions:

The 96h-LC50 was 0.65 mg/L based on nominal concentrations (95% confidence interval between 0.48 and 0.99 mg/L). When expressed in terms of calculated average exposure concentrations the 96h-LC50 was 0.40 (95% confidence interval between 0.27 and 0.58 mg/L). The LC50 values were reached after 24 hours of exposure.

Description of key information

The 96h-LC50 was 0.65 mg/L based on nominal concentrations (95% confidence interval between 0.48 and 0.99 mg/L). When expressed in terms of calculated average exposure concentrations the 96h-LC50 was 0.40 mg/L (95% confidence interval between 0.27 and 0.58 mg/L). The LC50 values were reached after 24 hours of exposure.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

0.4 mg/L