Reaction product of 2-hydroxybenzoic acid, styrene and oxozinc

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:

2012-10-25 to 2013-03-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 9.9, 14.8, 22.2, 33.3 and 50 mg/L.
- Sampling method: Samples were taken from the test concentration and the control at the start and at the end* of the experiment and analysed by a reverse phase HPLC method using UV detection.

Vehicle:

no

Details on test solutions:

Fish were held prior to the study in reconstituted water (ISO medium, prepared according to recommendation of Annex 2 of the OECD 203 guideline).
Separate stock solutions of individual substances were first prepared in deionised water. The ISO medium was prepared by adding 25 mL from each of four stock solutions to one litre deionised water.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

Origin:
The test fish were bred and held in Fish Laboratory of TOXI-COOP Zrt. (8237 Tihany, Klebelsberg K. u. 3.). The fish used as breeding stock for production of test animals obtained from a commercial source and held in the laboratory since 08 July 2010.
Number of animals:
There were 7 animals at the test concentrations and 10 animals in the control group (no parallels were used).
Age and size of animals:
Juveniles were used. The body length of each fish used was within the range of 2 ± 1 cm.
The mean body weight of the test animals per groups was recorded on the day 0 (at the start of the test). The loading of the test aquaria was calculated (g fish / liter test solution). The loading did not exceed 1.0 g/L.
Sex:
Both female and male (were not separated).
Animal health:
Apparently healthy animals were used in the test.
Acclimatization, holding prior to the study:
Since fish used in the test were bred in the laboratory under similar conditions (water temperature, pH, oxygen concentration etc.) and in the same type of water as that used in the experiment, no acclimatization period was necessary prior to start of the test. Fish were held according to the relevant SOPs of TOXI-COOP ZRT. During holding, fish were fed with appropriate, commercial diet for fish at least three times per week until one day before the test start. The health of the breeding was continuously monitored and any mortality or abnormal behaviour recorded. There was no mortality occurred in seven days before the start of experiment, therefore the batch was considered to be acceptable for testing.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Hardness:

The reconstituted water (ISO medium) has an approximate theoretical total hardness of 249 mg/L (as CaCO3).

Test temperature:

The aquariums were kept in a temperature-controlled tray. The measured temperatures were within a range of 22.2 – 23.4 °C in the test aquariums and were constant (the maximum deviation did not exceed ± 2 °C).

pH:

The test was carried out without adjustment of pH. The measured pH values were in the range of 7.42 – 7.86 during the test.

Dissolved oxygen:

The dissolved oxygen concentration was in the range of 75.8 – 97.9 % of the air saturation value at the temperature used. Test solutions were not aerated during the test.

Nominal and measured concentrations:

The following nominal concentrations were used in the main study: 9.9, 14.8, 22.2, 33.3 and 50 mg/L.
The corresponding measured test item concentrations (calculated as the geometric mean of the measured start and end values) were: 9.8, 14.1, 22.1, 33.3 and 50.1 mg/L.

Details on test conditions:

- Results used to determine the conditions for the definitive study:

Reference substance (positive control):

no

Duration:

96 h

Dose descriptor:

EC100

Effect conc.:

50.1 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

96 h

Dose descriptor:

LOEC

Effect conc.:

33.3 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

22.1 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

29.3 mg/L

95% CI:

21.6 - 33.9

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

Five different concentrations arranged in a geometric series with a separation factor of 1.5 and a concurrent control were tested in the main experiment. The nominal test item concentrations were 9.9, 14.8, 22.2, 33.3 and 50 mg/L.
The measured concentrations of the test item were 9.52, 14.0, 21.7, 32.6 and 50.0 mg/L at the start and 10.06, 14.2, 22.5, 34.0 and 50.2* mg/L at the end of the test.
* Remark: The end concentration of the highest concentration (50 mg/L nominal) was analytically determined after 24h exposure when all fish were observed as dead.
The exposure concentrations were calculated as the geometric mean of start and end values and were 9.8, 14.1, 22.1, 33.3 and 50.1 mg/L.
All biological results are based on the measured geometric mean concentrations.


All fish were observed as dead at the highest tested concentration (50.1 mg/L) after 24 hours of exposure. Reduced activity and staying at the bottom of the aquarium was observed in the case of five fish at the 3 and 6-h observation period.
In the concentration of 33.3 mg/L six fish were noticed as dead by the end of the test. Reduced activity and staying at the bottom of the aquarium was observed in the fish remained alive during the test from the 24-h observation period. At the 3 and 6-h observation period these clinical symptoms were detected in three fish (from 7) only.
In the concentrations of 9.8, 14.1, 22.1** mg/L and in the control mortality and sub-lethal effects were not observed during the experiment.
** Remark: At the concentration of 22.1 mg/L similar clinical symptoms were noticed as in the two highest concentration (33.3 and 50.1 mg/L) at the 3 and 6-h observation period, however from the 24h to 96h period all fish were found to be normal.
The LC50 values of the test item and their confidence limits were calculated using Probit analysis by SPSS PC+ software. The NOEC, LOEC and LC100 were determined directly from the raw data.
Biological results are related to the geometric mean of the measured test item concentrations during the test.

Validity criteria fulfilled:

yes

Conclusions:

In this 96-hour acute toxicity test with Zebrafish (Brachydanio rerio) the 96 h LC50 value was determined to be 29.3 mg/L, and the 96 h NOEC was determined as 22.1 mg/L. The results are based on the measured geometric mean concentrations.

Executive summary:

In a 96-hour acute toxicity test according to OECD 203 TG under GLP compliance, young Zebrafish (Brachydanio rerio)were exposed  to aqueous test media containing the test item for 96 hours at a range of concentration in a static test system  to evaluate the influence of the test item Reaction product of 2-hydroxybenzoic acid, styrene and oxozinc on zebrafish (Brachydanio rerio).Test concentrations were prepared by appropriate diluting of a saturated stock solution.
The following nominal concentrations were used in the main study: 9.9, 14.8, 22.2, 33.3 and 50 mg/L.
The corresponding measured test item concentrations (calculated as the geometric mean of the measured start and end values) were: 9.8, 14.1, 22.1, 33.3 and 50.1 mg/L.

All fish were observed as dead at the highest tested concentration (50.1 mg/L) after 24 hours of exposure. Reduced activity and staying at the bottom of the aquarium was observed in the case of five fish at the 3 and 6-h observation period.
In the concentration of 33.3 mg/L six fish were noticed as dead by the end of the test. Reduced activity and staying at the bottom of the aquarium was observed in the fish remained alive during the test from the 24-h observation period. At the 3 and 6-h observation period these clinical symptoms were detected in three fish (from 7) only.
In the concentrations of 9.8, 14.1, 22.1 mg/L and in the control mortality and sub-lethal effects were not observed during the experiment.

The 96 h LC50 value was determined to be 29.3 mg/L, and the 96 h NOEC was determined as 22.1 mg/L. The results are based on the measured geometric mean concentrations.

Description of key information

The 96 h LC50 value was determined to be 29.3 mg/L, and the 96 h NOEC was determined as 22.1 mg/L. The results are based on the measured geometric mean concentrations.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

LC50

Effect concentration:

29.3 mg/L

Additional information

In a 96-hour acute toxicity test according to OECD 203 TG under GLP compliance, young Zebrafish (Brachydanio rerio)were exposed  to aqueous test media containing the test item for 96 hours at a range of concentration in a static test system  to evaluate the influence of the test item Reaction product of 2-hydroxybenzoic acid, styrene and oxozinc on zebrafish (Brachydanio rerio).Test concentrations were prepared by appropriate diluting of a saturated stock solution.
The following nominal concentrations were used in the main study: 9.9, 14.8, 22.2, 33.3 and 50 mg/L.
The corresponding measured test item concentrations (calculated as the geometric mean of the measured start and end values) were: 9.8, 14.1, 22.1, 33.3 and 50.1 mg/L.

All fish were observed as dead at the highest tested concentration (50.1 mg/L) after 24 hours of exposure. Reduced activity and staying at the bottom of the aquarium was observed in the case of five fish at the 3 and 6-h observation period.
In the concentration of 33.3 mg/L six fish were noticed as dead by the end of the test. Reduced activity and staying at the bottom of the aquarium was observed in the fish remained alive during the test from the 24-h observation period. At the 3 and 6-h observation period these clinical symptoms were detected in three fish (from 7) only.
In the concentrations of 9.8, 14.1, 22.1 mg/L and in the control mortality and sub-lethal effects were not observed during the experiment.

The 96 h LC50 value was determined to be 29.3 mg/L, and the 96 h NOEC was determined as 22.1 mg/L. The results are based on the measured geometric mean concentrations.