5-bromo-5-nitro-1,3-dioxane

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:

25 Oct 2018 to 25 Jan 2019

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:

yes

Remarks:

the deviation had no effect on the outcome or overall integrity of the study.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identification: 5-Bromo-5-nitro-1,3-dioxane
Batch (Lot) Number: 201807021
Expiry date: 30 Jun 2020 (expiry date) (taken from label)
Physical Description: White crystal powder
Storage Conditions: At room temperature

Analytical monitoring:

yes

Details on sampling:

Sampling for Analysis of Test Concentrations
Samples for possible analysis were taken from all test concentrations and the control according to the schedule below. The method of analysis is described in the appended Analytical Report (Appendix 2).
Frequency 1. At the start of the test, after 48 hours and after 72 hours from the freshly prepared solutions.
2. At the first renewal (t=24h), second renewal (t=48h) and at the end of the test from the 24-hour old solutions.
Volume 1.6 mL from the approximate centre of the test vessels
Storage Samples were stored in a freezer (≤-15°C) until analysis at the analytical laboratory of the Test Facility
Additionally, reserve samples of 1.6 mL were taken from all test solutions for possible analysis. If not already used, these samples were stored in a freezer (≤-15°C) for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis.

Vehicle:

yes

Remarks:

Test medium without test item or other additives.

Details on test solutions:

The batch of 5-Bromo-5-nitro-1,3-dioxane tested was a white crystal powder with a purity of 99.63% and was completely soluble in test medium at the concentrations tested. No correction was made for the purity/composition of the test item. Preparation of test solutions was performed under dimmed light conditions and glassware used was wrapped in aluminium foil to minimize exposure to light.
In the range-finding test, preparation of test solutions started with the highest concentration of 100 mg/L applying an 11-minute period of ultrasonic treatment followed by an overnight period of magnetic stirring to completely dissolve 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.
In the final test, preparation of test solutions started with a stock solution of 5.0 mg/L in test medium by applying 10 – 11 minutes of ultrasonic treatment followed by an overnight period of magnetic stirring. During preparation of the stock for the third renewal after 72 hours of exposure, a subsequent 21 minutes of ultrasonic treatment was applied to completely dissolve the test item in test medium. The preparation procedure for the stock used for renewal after 48 hours of exposure was slightly adjusted. A 21-minute period of ultrasonic treatment followed by 1.25 hours of magnetic stirring was applied. This proved to be sufficient to obtain complete dissolution of test item.
Test concentrations were prepared by subsequent dilution of the stock solution in test medium and all solutions were observed to be clear and colorless at the end of the preparation procedure. Any residual volumes were discarded.

Test organisms (species):

Cyprinus carpio

Details on test organisms:

Species Carp (Cyprinus carpio, Teleostei, Cyprinidae) Linnaeus, 1758
Source ‘De Haar Vissen’, Zodiac proefacc. Wageningen University and Research Centre, The Netherlands.
Mean length Range-finding test: 3.5 ± 0.4 cm
Final test: 2.5 ± 0.1 cm
Mean weight1 Range-finding test: 0.48 ± 0.20 g
Final test: 0.19 ± 0.06 g
Characteristics F1 from a single parent-pair bred in UV-treated water.
Reason for selection This system has been selected as an internationally accepted species.
Total fish used 54
1- Ten fish of the batch used for the test, were weighed and measured prior to the start of the test.

Holding
Quarantine/Acclimatisation: At least 12 days after delivery.
Holding Medium Adjusted ISO medium (see section 4.8.2.2).
Measurements Conductivity, pH, nitrate, nitrite and ammonia concentration: once a week. Temperature was continuously recorded. In addition, pH and temperature were measured before transferring the fish to the test system.
Water quality parameters Were kept within the optimum limits for the respective fish species.
Feeding Daily with pelleted fish food (Essence 300-500 µm, Alltech Coppens International B.V., Helmond, The Netherlands)
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:

The hardness of test medium expressed as CaCO3: 180 mg/L.

Test temperature:

temperature 20-24°C

pH:

pH: 6.0-8.5, constant within 1 unit

Dissolved oxygen:

oxygen > 60% of air saturation

Salinity:

Test medium
Adjusted ISO medium. The following salts (analytical grade) were added to tap water purified by Reverse Osmosis (RO-water, GEON Waterbehandeling, Berkel-Enschot, The Netherlands):

CaCl2.2H2O 211.5 mg/L
MgSO4.7H2O 88.8 mg/L
NaHCO3 46.7 mg/L
KCl 4.2 mg/L

Nominal and measured concentrations:

Measured concentrations
Samples taken from all test concentrations and the control were analysed. The measured concentrations in the freshly prepared and 24-hour old solutions were at the level of nominal throughout the test, i.e. ranged between 80 and 120% relative to nominal concentrations throughout the test. It should be noted that small test item responses were found in the control samples, however, were most likely caused by carry over since similar responses were measured in the blank Quality Control (QC) samples.
Additional samples taken from the freshly prepared solutions upon renewal after 48 hours of exposure were analysed in order to assess the effect of the slightly adjusted preparation procedure. Analysis revealed that the measured concentrations ranged between 103-121% relative to nominal concentrations. Hence, it can be stated that the adjusted preparation procedure had no effect on the test item concentrations in test medium.
It should be noted that the measured concentration was slightly above the criterion of 80 120% relative to nominal (i.e. 121%) in the fresh solution prepared at nominally 0.060 mg/L used for renewal after 48 hours of exposure. Nevertheless, no effects were recorded at this test concentration and thus was not used in the calculation of effect parameters.
Based on the obtained results, effect parameters were expressed in terms of analytically confirmed nominal concentrations.

Nominal concentrations
Seven fish per group were exposed to an untreated control and to nominal concentrations of 0.060, 0.12, 0.24, 0.48 and 0.96 mg/L.

Measured concentrations
. The measured concentrations in the freshly prepared and old solutions of the first and last 24-hour renewal interval were at the level of nominal, i.e. ranged between 80 and 120% relative to nominal concentrations throughout the 24-hour renewal intervals. Based on these results, effect parameters were expressed in terms of analytically confirmed nominal concentrations.

Details on test conditions:

All test conditions remained within the ranges prescribed by the study plan (pH: 6.0-8.5, constant within 1 unit; temperature 20-24°C, constant within 2°C; oxygen > 60% of air saturation).

Reference substance (positive control):

not specified

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

ca. 0.68 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

Range-Finding test
No mortality was observed at the lowest concentration of nominally 0.10 mg/L throughout the test. After three hours of exposure, fish exposed to 1.0 mg/L were observed to be swimming at the bottom of the tank, which was different from the behaviour of fish at the lowest test concentration. All fish exposed to nominally 10 and 100 mg/L were found dead after three hours of exposure, while all fish exposed to 1.0 mg/L died during the first 24 hours of the test.

Based on these results samples taken from nominally 0.10 and 1.0 mg/L were analysed. The measured concentrations at the start of the test were 0.078 and 1.0 mg/L, respectively, and decreased to 72 and 55% of the initial concentrations throughout the 96 hour exposure period
(Cumulative Mortality 3h-96h)
Nominal conc Initial no of fish ( 3h 24h 48h 72h 96h) Total mortality %
0.10 3 0 0 0 0 0 0
1.0 3 0 3 3 3 3 100
10 3 3 3 3 3 3 100
100 3 3 3 3 3 3 100

Mortality and Clinical Effects
No mortality was observed in the control and at the four lowest test concentrations throughout the test, while all fish exposed to nominally 0.96 mg/L were found dead after 24 hours of exposure. No clinical effects other than mortality were observed at any test concentration throughout the test.
The responses recorded in this test allowed for reliable determination of an LC50.

Experimental Conditions
All test conditions remained within the ranges prescribed by the study plan (pH: 6.0-8.5, constant within 1 unit; temperature 20-24°C, constant within 2°C; oxygen > 60% of air saturation).

Results with reference substance (positive control):

This reference test was carried out to check the sensitivity of the test system as used by Charles River Den Bosch. The reference item was pentachlorophenol (PCP, SIGMA, Art. P2604, Batch MKBW8316V).
Concentrations: 0.10, 0.22 and 0.46 mg/L in ISO-medium.

During the test the pH, oxygen concentration and the temperature of the medium were within the optimal ranges for fish.
Under the conditions of the present test, PCP induced no lethal effects in carp at 0.10 mg/L. The 96h-LC50 for carp exposed to PCP was 0.15 mg/L (95 % confidence interval between 0.10 and 0.22 mg/L). This effect was already reached within 24 hours of exposure.
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:

The LC50-values could not be determined using the maximum likelihood estimation method with the probits of the percentages of dead fish as function of the logarithms of the corresponding concentrations. 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.

Validity criteria fulfilled:

yes

Conclusions:

In conclusion, the 96h-LC50 for carp (Cyprinus carpio) exposed to 5-Bromo-5-nitro-1,3-dioxane was 0.68 mg/L based on analytically confirmed nominal concentrations (95% confidence interval between 0.48 and 0.96 mg/L) and was already reached after 24 hours of exposure.

Executive summary:

The objective of the study was to evaluate 5-Bromo-5-nitro-1,3-dioxane for its ability to generate acute toxic effects in Cyprinus carpio during an exposure period of 96 hours and, if possible, to determine the LC₅₀at all observation times.

The study procedure described in this report was based onthe 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 5-Bromo-5-nitro-1,3-dioxane tested was a white crystal powder with a purity of 99.63% and was completely soluble in test medium at the concentrations tested.Preparation of test solutions was performed under dimmed light conditions and glassware used was wrapped in aluminium foil to minimize exposure to light.

A semi-static final test was performed based on the results of a static exposure range-finding test. Seven fish per group were exposed to an untreated control and to nominal concentrations of 0.060, 0.12, 0.24, 0.48 and 0.96 mg/L. Since the test item is light sensitive, the exposure was performed under dimmed light conditions. The total exposure period was 96 hours and test solutions were renewed in 24-hour intervals. 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. The measured concentrations in the freshly prepared and old solutions of the first and last 24-hour renewal interval were at the level of nominal, i.e. ranged between 80 and 120% relative to nominal concentrations throughout the 24-hour renewal intervals. Based on these results, effect parameters were expressed in terms of analytically confirmed nominal concentrations. 

No mortality was observed in the control and at the four lowest test concentrations throughout the test, while all fish exposed to nominally 0.96 mg/L were found dead after 24 hours of exposure. No clinical effects other than mortality were observed at any test concentration throughout the test.

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

In conclusion,the 96h-LC₅₀was 0.68 mg/L based on analytically confirmed nominal concentrations (95% confidence interval between 0.48 and 0.96 mg/L) and was already reached after 24 hours of exposure.

Description of key information

The 96h-LC50 for carp (Cyprinus carpio) exposed to the test substance was 0.68 mg/L based on analytically confirmed nominal concentrations (95% confidence interval between 0.48 and 0.96 mg/L) and was already reached after 24 hours of exposure.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

0.68 mg/L