2-(2-thienyl)ethyl toluene-p-sulphonate

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 Oct 2017 - 10 Nov 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

2004

Deviations:

no

Test guideline 2

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

Test material

Test material information

Constituent 1

Reference substance name:

2-(2-thienyl)ethyl toluene-p-sulphonate

EC Number:

254-911-5

EC Name:

2-(2-thienyl)ethyl toluene-p-sulphonate

Cas Number:

40412-06-4

Molecular formula:

C13H14O3S2

IUPAC Name:

2-(thiophen-2-yl)ethyl 4-methylbenzene-1-sulfonate

Test material form:

solid: particulate/powder

Details on test material:

- Physical appearance: white to off-white powder
- Test item storage: at room temperature

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Control and all test concentrations
- Sampling method: 2.0 mL from the approximate centre of the test vessels, at t=0 h and t=48 h.
- Sample storage conditions before analysis: Samples were stored in a freezer (≤ -15°C) until analysis at the analytical laboratory of the Test Facility.
- At the end of the exposure period, the replicates were pooled at each concentration before sampling.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Direct addition to test medium. A test stock solution was prepared at a nominal loading rate of 100 mg/L, applying a three-day period of magnetic stirring to ensure maximum dissolution of the test item in medium. Thereafter, the aqueous Saturated Solution (SS) was collected by means of filtration through a 0.45 µm membrane filter (RC55, Whatman) and used as the highest test concentration. Lower test concentrations were prepared by subsequent dilutions of the SS in test medium. All test solutions were clear and colorless at the end of the preparation procedure.
- Controls: Test medium without test item or other additives.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Strain/clone: Straus 1820
- Source: In-house laboratory culture with a known history
- Age of parental stock: >2 weeks old
- Age at test initiation: <24 hours
- Validity of batch: Daphnids originated from a healthy stock, 2nd to 5th brood, showing no signs of stress such as mortality >20%, presence of males, ephippia or discoloured animals and there was no delay in the production of the first brood.

CULTIVATION
- Start of each batch: With newborn daphnids, i.e. less than 3 days old, by placing about 250 of them into 5 litres of M7 medium in an all-glass culture vessel.
- Maximum age of the cultures: 4 weeks
- Renewal of the cultures: After 7 days of cultivation half of the medium twice a week.
- Temperature of medium: 18-22°C
- Feeding: Daily, a suspension of fresh water algae.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test conditions

Hardness:

180 mg/L (as CaCO3)

Test temperature:

19 - 21°C

pH:

At t=0 h: 7.8-8.0
At t=48 h: 8.0-8.1

Dissolved oxygen:

At t=0 h: 8.7-9.7 mg/L
At t=48 h: 9.1-9.9 mg/L

Nominal and measured concentrations:

Nominal: 4.6, 10, 22, 46 and 100% of a SS prepared at a nominal loading rate of 100 mg/L
Measured concentrations decreased to 79-80% of initial during the exposure period. Therefroe, effect parameters were based on the average exposure concentrations (geometric mean of measured concentrations). See Table 1 and 2 in 'Any other information on results' for details on measured concentrations throughout the test.

Details on test conditions:

TEST SYSTEM
- Test vessel: 60 mL, all-glass, containing 50 mL of test solution
- Aeration: no aeration
- No. of organisms per vessel: 5 daphnids
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Adjusted ISO medium, prepared with tap water purified by Reverse Osmosis.
- Culture medium different from test medium: Yes
- Intervals of water quality measurement: pH and dissolved oxygen: At the beginning and at the end of the test, for all concentrations and the control. In addition, after 24 hours, immediately after counting the immobilized daphnids oxygen levels were measured in the test container with the solution of lowest concentration at which all the Daphnia magna were immobilized.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hours photoperiod daily

EFFECT PARAMETERS MEASURED
- Immobility (including mortality): at 24 hours and at 48 hours

RANGE-FINDING STUDY
- Test concentrations: Control, 1.0, 10 and 100% of SS prepared at nominal loading rate of 100 mg/L
- Results used to determine the conditions for the definitive study: yes; expected EC50 was between 10 and 100% of SS prepared at nominal loading rate of 100 mg/L.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate (performed Sep 2017)

Results and discussion

Effect concentrations

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

2.9 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mobility

Remarks on result:

other: 95% confidence interval: 2.4-3.1 mg/L

Details on results:

- Behavioural abnormalities: none observed in the Control group.
- Mortality of control: none
- No immobility was observed in the control and the two lowest test concentrations. At the end of the test a steep dose-response curve was observed at the three highest concentrations tested. See Table 3 in 'Any other information on results' for full details on immobility.
- All water quality parameters remained within the requirements as laid down in the study plan throughout the test.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- The 24h-EC50 was 0.59 mg/L with a 95% confidence interval between 0.52 and 0.68 mg/L.
- The 48h-EC50 was 0.41 mg/L with a 95% confidence interval between 0.34 and 0.46 mg/L.
- The responses in this reference test with K2Cr2O7 are within the ranges of the expected responses at the different concentrations, i.e. the 48h-EC50 was between 0.3 and 1.0 mg/L. Hence, the sensitivity of this batch of D. magna was in agreement with the historical data collected at the Test Facility.

Reported statistics and error estimates:

The 24 and 48h-EC50-value were calculated from the probits of the percentages of affected daphnids and the logarithms of the corresponding test item concentrations (average exposure) using the maximum likelihood estimation method.
ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used to perform the analyses.

Any other information on results incl. tables

Table 1: Final Test: Test Samples

Time of sampling [hours]	Percentage of SS^(a) [%]	Analyzed concentration [mg/L]	Relative to initial [%]
0	0	n.d.
	4.6	0.468
	10	1.01
	22	2.23
	46	4.63
	100	10.2
48	0	n.d.	n.a.
	4.6	0.373	80
	10	0.809	80
	22	1.77	79
	46	3.73	80
	100	8.15	80

Samples were stored in the freezer (≤ -15°C) until the day of analysis.

(a)  Percentage of a saturated solution (SS) prepared at a loading rate of 100 mg/L.

n.d. Not detected.

n.a. Not applicable.

Table 2: Average Exposure Concentration Versus Nominal Concentration

%SS prep. at a loading rate of 100 mg/L	Measured concentrations (mg/L)		Average exposure conc. (mg/L)
	t=0h	t=48h
4.6	0.47	0.37	0.42
10	1.0	0.81	0.91
22	2.2	1.8	2.0
46	4.6	3.7	4.2
100	10	8.2	9.1

Table 3: Number of Introduced Daphnids and Incidence of Immobility in the Final Test

Time (h)	Replicate	Average exposure concentration (mg/L)
		Control	0.42	0.91	2.0	4.2	9.1
0	A	5	5	5	5	5	5
	B	5	5	5	5	5	5
	C	5	5	5	5	5	5
	D	5	5	5	5	5	5
	Total introduced	20	20	20	20	20	20
24	A	0	0	0	0	5(1)^#	5*
	B	0	0	0(1)	0	4	5
	C	0	0(1)	0	0	5	5
	D	0	0	0	0	5	5
	Total immobilised	0	0	0	0	19	20
	Effect %	0	0	0	0	95	100
48	A	0	0	0	0	5	5
	B	0	0	0	0	5	5
	C	0	0	0	0	5	5
	D	0	0	0	1	5	5
	Total immobilised	0	0	0	1	20	20
	Effect %	0	0	0	5	100	100

( ) between brackets: number of daphnia observed trapped at the surface of the test solutions. These organisms were re-immersed into the respective solutions before recording of mobility

^# Microscopic observation revealed no test item attached to the daphnids
* Measured oxygen was 9.4 mg/L

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

See 'Overall remarks' for details on validity criteria.

Conclusions:

The 48h-EC50 for Daphnia magna exposed to the test item was 2.7 mg/L based on geometric mean measured concentrations (95% confidence interval between 2.4 and 3.1 mg/L).

Executive summary:

In a 48 h acute toxicity test conducted according to OECD guideline 202, Daphnia magna were exposed to an untreated control and 4.6, 10, 22, 46 and 100% test solutions of a Saturated Solution prepared at a nominal loading rate of 100 mg/L (4 replicates with 5 daphnids per replicate for each test group). Measured concentrations decreased to 79 -80% of initial, therefore effect parameters were based on geometric mean measured concentrations. No immobility was observed in the control and the two lowest test concentrations. At the end of the test a steep dose-response curve was observed at the three highest concentrations tested.

The 48 -hEC50 for immobility was 2.7 mg/L (95% confidence interval 2.4 -3.1 mg/L), based on geometric mean measured concentrations. The study is considered to be reliable without restrictions.