S-2-Benzothiazolyl (Z)-2-(2-aminothiazol-4-yl)-2-acetyloxyiminothioacetate

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

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Guideline study with GLP. The study is well performed, except for using a loading rate of 100 mg of the insoluble test substance, and not a concentration at the solubility limit. Thereby mainly the enriched impurities of the substance were tested. A Klimisch 2 is allocated.

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

Actual concentrations of the test substance in the test media were determined immediately before placing the fish in the test vessels and then after 4 hours.

Vehicle:

no

Details on test solutions:

According to the scheme in Section 4.8 'Water solubility' a substance is classified as 'insoluble' if the solubility in water is <0.1 mg/L. The water solubility of T15 -AE is 0.077 mg/L at 20 °C.
Test medium was used to prepare the test substance solution.
The test medium with 100 mg/L loading rate was prepared by weighing in 0.512 g test substance and filling up to 5 L dilution water. This suspension was treated for 30 minutes by ultrasonication at approximately 20 °C. A water accommodated fraction, obtained after filtration with a 0.21µm cellulose acetate filter, was then tested.
Dilution water: Local tap water was mixed 1:1 with deionized water to achieve the appropriate water hardness of 250 mg CaCO3/L, determined in the control vessel.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

Species: Zebrafish (Brachydanio rerio)
Supplier: Amazonas Aquarium, A-2840 Grimmenstein, Austria
Receipt of animals: 9 February 2005.
Length (cm): Approximately 2.5 cm (mean of the control group at the end of study).
Weight (g): ca. 0.3

Animal maintenance during acclimatisation and during exposure:
Water: Dilution water for holding and exposure
Aeration: Holding: continuous aeration by compressed air (water and oil filtered) and additional aeration by water recirculation using an EHEIM 2211 filter pump. Exposure: no aeration.
Temperature: Holding: ca. 20 °C. Exposure: 20 °C.
Tanks: Holding: full glass vessel (47 cm x 29 cm x 31 cm}, filled with ca. 40 L, covered with a transparent plastic lid. Water was purified by recirculation (power filter pump EHEIM 2211) through filter wool. Exposure: full glass vessels (29 cm x 20 cm x 24 cm}, filled with 5 litres of control or test medium, covered with transparent plastic lids.
Light: Artificial light from 4 a.m. to 8 p.m.
Feeding: Feed: TetraMin; Tetra Werke, Dr.rer.nat. Ulrich Baensch GmbH, D-49304 Melle. Holding: daily. Exposure: no feeding (last feeding about 24 hours before the start of the exposure).
Acclimatisation period: Approximately 9 weeks.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Post exposure observation period:

n.a.

Hardness:

250 mg CaCO3/L.
Local tap water was mixed 1:1 with deionized water to achieve the appropriate water hardness of 250 mg CaCO3/L, determined in the control vessel.

Test temperature:

20 °C

pH:

7.8 - 8.1 in the test group.
7.7 - 8.1 in the control group.

Dissolved oxygen:

80 to 97 % in the test group.
93 to 98 % in the control group.

Salinity:

n.a.

Nominal and measured concentrations:

Nominal loading rate: 100 mg/L.

Details on test conditions:

7 fish were used per group.

Reference substance (positive control):

not required

Duration:

24 h

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: Concentration refer to the loading rate.

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: Concentration refer to the loading rate.

Duration:

72 h

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: Concentration refer to the loading rate.

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: Concentration refer to the loading rate.

Duration:

96 h

Dose descriptor:

LC100

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: Concentration refer to the loading rate.

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: Concentration refer to the loading rate.

Details on results:

Mortality:
Negative control group: All animals survived until the end of the test.
Test substance group: All animals survived until the end of the test.

Animal observation:
All animals of the test substance group and of the negative control group were inconspicuous in behaviour and appearance until the end of the test.

Actual T15-AE concentration: At the start of theexposure the test substance concentration was 0.079 mg/L. Four hours after the start the test substance concentration was below the detection limit of the used method (0.0004 mg/L).

Results with reference substance (positive control):

n.a.

Reported statistics and error estimates:

n.a.

Sublethal observations / clinical signs:

Except for the maintenance of the test substance concentration all quality criteria were fulfilled:

• Constant conditions were maintained (temperature, pH).

• No mortality occurred in the negative control group.

• The dissolved oxygen concentration was higher than 60 % of the air saturation value throughout the test.

Validity criteria fulfilled:

yes

Remarks:

except for the

Conclusions:

The LC50,96h, based on the loading rate, is higher than 100 mg/L.

Executive summary:

The acute toxicity of T15-AE and its degradation products to Zebrafish (Brachydanio rerio) was determined in a 96 hours static test according to the EU-method C.1. The solubility of T15-AE in water is very low. Therefore an aqueous extract (loading rate 100 mg/L in dilution water) was prepared to achieve a dissolved test substance concentration as high as possible. After approximately 30 minutes of ultrasonic treatment and subsequent filtration (filter size 0.2 µm) the obtained water accommodated fraction was tested (limit test). 7 fish were used each for the test substance group and for one control group (dilution water only). By this procedure predominantly the enriched and possibly better water soluble impurities are tested.

Results:

No mortality and no adverse effects were noted in the test substance group and in the negative control group.

The actual T15 -AE concentration of the filtrate was 0.079 mg/L at the start of the test and declined rapidly within 4 hours to below the detection limit.

pH and temperature were stable throughout the test period. Dissolved oxygen concentration was always higher than 60 % of the air saturation value at the appropriate temperature.

The LC50,96h, based on the loading rate, is higher than 100 mg/L.

Description of key information

The LC50,96h, based on the loading rate, for Brachydanio rerio is higher than 100 mg/L. The LC50 is higher than the solubility of the substance in the medium of ca. 0.08 mg/L. The result is not directly suitable for a classification, but it demonstrates that the mixture, i.e. the pure substance and its impurities, as far as they are soluble at 100 mg mixture/L, is not aquatic toxic.

The impurity benzothiazole-2-thiol (typically 1.2 % in T15-AE) is classified in CLP as Aquatic Acute 1 H400 and Aquatic Chronic 1 H410. The LC50 of Benzothiazole-2-thiol = 0.73 mg/L, the water solubility is 118 mg/L at 25 °C and the substance is not readily biodegradable (results obtained from the ECHA website). Applying the additivity formula of section 4.1.3.5.2. in CLP, an EC50 of the mixture (=T15-AE) of 61 mg/L is calculated. This is in acceptable agreement with the experimentally obtained result.

An LC50 far above the solubility limit is confirmed by a QSAR-calculation leading to an LC50 of 42 or 189 mg/L, depending on the model used.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

61 mg/L