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

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

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 just above the solubility. Thereby mainly the enriched impurities of the substance were tested. A Klimisch 2 is allocated.

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

Actual concentrations of the test substance were determined before the test and after 48 h.

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.
The stock solutions for preparation of the reconstituted water according to ISO 6341 were obtained by MicroBioTests Inc. (Batch No. ISOD0300903). The water was aerated and the pH, the dissolved oxygen concentration and the water hardness were checked before using.
The test medium with 100 mg/L loading rate was prepared by weighing in 10.9 mg test substance and filling up to 100 mL reconstituted water. This suspension was treated for 30 minutes by ultrasonication at 20 °C. A water accommodated fraction, obtained after filtration with a 0.2 µm cellulose acetate filter, was then tested.

Test organisms (species):

Daphnia magna

Details on test organisms:

Neonates of Daphnia magna, less than 24 hours old, were obtained by hatching ephippia, supplied in the microbiotest "Daphtoxkit F TM" (MicroBioTests Inc., 9810 Nazareth, Belgium). Batch No. of the ephippia: DM200704.
The ephippia were hatched in a petri dish containing reconstituted water in a temperature controlled room at ca. 20 - 22 °C under continuous illumination of approximately 6000 Lux. According to MicroBioTests Inc. hatching occurs mostly between 72 and 80 hours after initiation of hatching. Collection of neonates should be latest at 90 h. Approximately 2 hours before beginning of the test the neonates were fed with a suspension of Spirulina powder, as also in traditional cultures the neonates have the opportunity to take up some feed particles. The neonates were collected for the test at approximately 87 hours after initiation of hatching.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Post exposure observation period:

n.a.

Hardness:

250 mg CaCO3/L

Test temperature:

20 °C.

pH:

7.6 - 7.8 in the control group.
7.6 - 7.9 in the test group.

Dissolved oxygen:

8.7 - 9.0 mg/L in the control group.
7.6 - 7.9 mg/L in the test group.

Salinity:

n.a.

Nominal and measured concentrations:

Nominal: 100 mg/L, based on the loading rate.
Actual, mean: 0.15 mg pure T15-AE/L at 0 h. Below the detection limit of 0.0004 mg/L at 48 h.

Details on test conditions:

A negative control group was used.
20 daphnids, divided into 4 replicates (5 daphnia each), were used for the test substance group and also for the control group.

Exposure technique:
Water: Reconstituted water.
Aeration: No extra aeration, but slight circulation of air is still possible when the lid of the test plate is closed.
Temperature: Ca. 21 °C, monitored daily in a "rinsing well".
Light: Artificial light from 4 a.m. to 8 p.m.
Feed: No feed.
Transfer to the test wells: The "rinsing wells" and the wells for the replicates of the test substance and the control respectively were filled with 10 mL test medium. For the test substance and for the control at least 20 actively swimming neonates were transferred with a micropipette from the hatching petri dish to the "rinsing well". Then 5 neonates from the "rinsing wells" were transferred to each of the 4 wells of the appropriate group.


 

Reference substance (positive control):

yes

Remarks:

performed periodically with Na-dichromate.

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: Concentration is based on the loading rate.

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: Concentration is based on the loading rate.

Duration:

48 h

Dose descriptor:

EC100

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: Concentration is based on the loading rate.

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: Concentration is based on the loading rate.

Details on results:

• No immobilisation was noted in the test.
• The concentration of the pure T15 -AE declined from initially 0.152 mg/L to below the detection limit of 0.0004 mg/L.
• Temperature was stable during the test period.
• Dissolved oxygen concentrations in the test vessels were not lower than 8.0 mg/L at each time determined (EC guideline: higher than 3 mg/L).
• pH of the test substance solution was between 7.6 and 7.9 (EC guideline: deviation of not more than one unit).


Quality criteria
Except for the maintenance of the test substance concentration the quality criteria were fulfilled:
• Constant conditions were maintained (temperature, pH).
• There was no immobilisation in the negative control group.
• There were no control daphnia trapped at the water surface.
• The dissolved oxygen concentration was higher than 3 mg/L at each determination.

Results with reference substance (positive control):

EC50,24h = 1.19 mg/L.

Reported statistics and error estimates:

n.a.

Validity criteria fulfilled:

yes

Conclusions:

The EC50,48h, based on the loading rate, is higher than 100 mg/L.

Executive summary:

The acute toxicity of the substance to Daphnia magna was determined in a 48 hours static test according to the EC-method C.2. A limit test with 100 mg loading rate of the test substance/L was performed. By this procedure predominantly the enriched, possibly better water soluble impurities are tested.

Neonates of Daphnia magna, hatched from ephippia and not more than 24 hours old, were exposed. One negative control group was exposed to reconstituted water only. 20 daphnids, were used for the test substance group and also for the control group.

Results:

• No immobilisation was noted in the test.

• Immobilisation of control group daphnids was 0 % at the end of the test

• There were no daphnids trapped at the water surface.

• The concentration of the pure T15 -AE declined from initially 0.152 mg/L to below the detection limit of 0.0004 mg/L.

• Temperature was stable during the test period.

• Dissolved oxygen concentrations in the test vessels were not lower than 8.0 mg/L at each time determined (EC guideline: higher than 3 mg/L).

• pH of the test substance solution was between 7.6 and 7.9 (EC guideline: deviation of not more than one unit).

The EC50,48h, based on the loading rate, is higher than 100 mg/L.

Description of key information

The EC50,48h for daphnids, based on the loading rate, is higher than 100 mg/L. The EC50 is higher than the solubility of the substance in the medium of ca. 0.15 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 EC50 of Benzothiazole-2-thiol = 0.71 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 59 mg/L is calculated. This is in acceptable agreement with the experimentally obtained result.

An EC50 above the solubility limit is confirmed by a QSAR-calculation leading to an EC50 is 3.2 or 112 mg/L, depending on the model used.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

59 mg/L