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

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 Oct 2017 - 16 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 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

2006 (Annex 5 corrected 28 July 2011)

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, at t=0 h, t=24 h and t=72 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 with algae were pooled at each concentration before sampling.
- Compliance with the quality criteria regarding maintenance of actual concentrations was checked by running a test vessel at an intermediate item concentration but without algae and samples for analysis were taken at the start, after 24 hours of exposure and at the end of the test period.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Test solutions were prepared at a nominal loading rate of 100 mg/L by 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 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.
- After preparation, volumes of 50 mL were added to each replicate of the respective test concentration. Subsequently, 1 mL of an algal suspension was added to each replicate providing a cell density of 10^4 cells/mL.
- Controls: Test medium without test item or other additives

Test organisms

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: NIVA CHL 1
- Source: In-house laboratory culture
- Age of inoculum (at test initiation): 3 days
- Method of cultivation: Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24°C.
- Pre-culture: 3 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1 x 10^4 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.
- Stock culture medium: M1, according to the NPR 6505 (Nederlandse Praktijk Richtlijn no. 6505).
- Pre-culture medium and test medium: M2, according to OECD 201.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Hardness:

0.24 mmol/L (24 mg CaCO3/L)

Test temperature:

22 - 23°C

pH:

At t=0 h: 8.0-8.1
At t=72 h: 7.7-8.3

Nominal and measured concentrations:

Nominal: Solutions containing 0.32, 1.0, 3.2, 10, 32 and 100% of a Saturated Solution prepared at a loading rate of 100 mg/L.
Measured: 0.024, 0.074, 0.24, 0.71, 2.4, 7.2 mg/L (Time Weighted Average concentrations). Time Weighted Average concentrations were calculated to determine the effect parameters because concentrations were found to decrease with time. See Tables 1 and 2 in 'Any other information on results' for details on measured concentrations and TWA.

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL, all-glass, containing 50 mL of test solution
- Capped vessels were distributed at random in the incubator and daily repositioned. During incubation the algal cells were kept in suspension by continuous shaking
- Initial cells density: 1 x 10^4 cells/mL
- Control end cells density (mean): 275 x 10^4 cells/mL
- 3 replicates of each test concentration
- 6 replicates of the control
- 1 extra replicate of each test group for sampling purposes
- 1 or 2 replicates of each test concentration without algae


GROWTH MEDIUM
- Standard medium used: yes, M2 medium

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: M2, formulated using Milli-RO water (tapwater purified by reverse osmosis)
- Intervals of water quality measurement: pH: at the beginning and at the end of the test for all test concentrations and the control. Temperature of medium: continuously in a temperature control vessel.

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod, light intensity and quality: Continuously using TLD-lamps with a light intensity at 87 μE/m2/s

EFFECT PARAMETERS MEASURED
- Cell density was measured at t= 24, 48, and 72 h.
- Determination of cell concentrations: At the beginning of the test, cells were counted using a microscope and a counting chamber. Thereafter, cell densities were determined by spectrophotometric measurement of samples at 680 nm using a spectrophotometer with immersion probe (pathlength =10 mm). Algal medium was used as blank and the extra replicates, without algae, as background for the treated solutions.

TEST CONCENTRATIONS
- Combined Limit/range finding study test concentrations: Control, 1.0, 10 and 100% of SS prepared at a nominal loading rate of 100 mg/L
- Results used to determine the conditions for the definitive study: yes; expected ErC50 was between 10 and 100% of SS prepared at a nominal loading rate of 100 mg/L

Reference substance (positive control):

yes

Remarks:

Potassium dichromate (performed Sep 2017)

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

3.2 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Remarks on result:

other: 95% confidcence interval: 3.1-3.3 mg/L

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

1.1 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence interval: 0.98-1.2 mg/L

Effect concentrations 3

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.074 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Remarks on result:

other: Based on statistical significance

Effect concentrations 4

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.24 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Remarks on result:

other: Based on biological relevance

Details on results:

- Exponential growth in the control: yes
- Observation of abnormalities: Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to 0.71 mg/L (TWA concentration) when compared to the control.
- Growth rates were in the range of the controls at the two lowest test concentrations during the 72-hour test period, whereas the growth rates of algae exposed to TWA concentrations of 0.24 mg/L and higher were increasingly reduced. Statistically significant inhibition of growth rate was found at test concentrations of 0.24 mg/L and higher. However, the effects observed at 0.24 mg/L were considered not biologically relevant and therefore, the NOErC based on biological relevance was set to 0.24 mg/L.
- 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 EC50 for growth rate inhibition (72h-ErC50) was 1.1 mg/L with a 95% confidence interval ranging from 1.1 to 1.1 mg/L. The historical ranges for growth rate inhibition lie between 0.82 and 2.3 mg/L. Hence, the 72h-ErC50 for the algal culture tested corresponds with this range.

Reported statistics and error estimates:

Statistical significance: Multiple Sequentially-rejective Welsh-t-test After Bonferroni-Holm, α=0.05, one-sided, smaller.
ECx: Weibull analysis using linear max. likelihood regression.
The calculations were performed with ToxRat Professional v. 3.2.1. (ToxRat Solutions® GmbH, Germany).

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.
	0.32	0.031^(c)
	1.0	0.0947
	3.2	0.303
	3.2^(b)	0.311
	10	0.917
	32	2.94
	100	9.19
24	0	n.d.	n.a.
	0.32	0.026^(c)	85
	1.0	0.0839	89
	3.2	0.269	89
	3.2^(b)	0.275	89
	10	0.799	87
	32	2.59	88
	100	7.30	79
72	0	n.d.	n.a.
	0.32	0.017^(c)	55
	1.0	0.052^(c)	55
	3.2	0.162	54
	3.2^(b)	0.218	70
	10	0.516	56
	32	1.83	62
	100	6.08	66

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.

^(b)   Without algae.

^(c)   Estimated value, calculated by extrapolation of the calibration curve.

n.d. Not detected.

n.a. Not applicable.

Table 2: Measured Concentrations Versus Nominal Concentrations

Nominal conc. (mg/L)	Measured concentration (mg/L)			TWA (mg/L)
	t=0h	t=24h	t=72 h
0.32	0.031	0.026	0.017	0.024
1.0	0.0947	0.0839	0.052	0.074
3.2	0.303	0.269	0.162	0.24
3.2#	0.311	0.275	0.218	0.26
10	0.917	0.799	0.516	0.71
32	2.94	2.59	1.83	2.4
100	9.19	7.30	6.08	7.2

# – without algae.

Table 3: Growth Rate And Percentage Inhibition For The Total Test Period

TWA conc. (mg/L)	Mean	Std. Dev.	n	%Inhibition
Control	1.872	0.0103	6
0.024	1.875	0.0029	3	-0.1
0.074	1.874	0.0072	3	-0.1
0.24	1.788	0.0016	3	4.5^#
0.71	1.527	0.0274	3	18.4*
2.4	1.320	0.0109	3	29.5*
7.2	0.014	0.0247	3	99.2*

* - effect was statistically significant

^#- effect was statistically significant but biologically not relevant (<10%).

Table 4: Growth Rate And Percentage Inhibition At Different Time Intervals

TWA conc. (mg/L)	n	0 – 24 h		24 – 48 h		48 – 72h
		Mean	%Inhibition	Mean	%Inhibition	Mean	%Inhibition
Control	6	2.084	0.0	1.982	0.0	1.551	0.0
0.024	3	2.071	0.6	1.987	-0.2	1.566	-1.0
0.074	3	2.053	1.5	2.011	-1.5	1.557	-0.4
0.24	3	2.187	-5.0	1.666	16.0	1.512	2.5
0.71	3	1.913	8.2	1.500	24.3	1.168	24.7
2.4	3	1.680	19.4	1.216	38.7	1.065	31.3
7.2	3	0.000	100.0	0.357	82.0	-0.315	120.3

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

See 'Overall remarks' for details on validity criteria

Conclusions:

The 72-h ErC50, ErC10 and NOErC for Pseudokirchneriella subcapitata was respectively 3.2 mg/L, 1.1 mg/L and 0.24 mg/L (based on biological relevance), based on nominal concentrations.

Executive summary:

In a 72 h toxicity study conducted according to OECD guideline 201 and GLP principles, freshwater algae (Pseudokirchneriella subcapitata) were exposed to the test item at 0.32, 1.0, 3.2, 10, 32 and 100% of a Saturated Solution prepared at a nominal loading rate of 100 mg/L (3 replicates) and an untreated control (6 replicates). Measured concentrations at the end of the exposure period had decreased to 54 -70% of the initial measured concentrations, therefore the Time Weighted Average exposure concentrations were used to determine the effect parameters. At the end of the test, a dose-related increase of inhibition of growth rate was observed up to 99% inhibition at the highest test concentration.The EC50 for growth rate inhibition (72h-ErC50) was 3.2 mg/L, the 72h-ErC10 was 1.1 mg/L. The 72h-NOErC was 0.24 mg/L and 0.074 mg/L based on biological relevance and statistical significance, respectively. The study met all validity criteria, and is considered reliable without restriction.