Methyl toluene-4-sulphonate

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 September 2017 to 07 September 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

OECD Guideline for Testing of Chemicals, Section 2, No. 201: "Freshwater Alga and Cyanobacteria, Growth Inhibition Test", adopted 28th July, 2011.

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II) (January 2012)

Version / remarks:

EPA Ecological Effects Test Guidelines, OCSPP 850.5400, Algal Toxicity, Tiers I and II, EPA 712-C-96-164, April 1996.

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

Commission Regulation (EU) 2017/735 of 14 February 2017 amending, for the purpose of its adaptation to technical progress, the Annex to Regulation (EC) No 440/2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH), Annex IV Part C, C.3

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

No further details specified in the study report.

Analytical monitoring:

yes

Details on sampling:

Analytical measurement was performed at the applied test concentration levels and from the control at the beginning of the test and 24 hour intervals thereafter during the experiment.
The samples were analysed by HPLC system.

Vehicle:

no

Details on test solutions:

A stock solution with a nominal concentration of 100.0 mg/L was prepared with the test item and algal growth medium (OECD Medium) using ultrasonic bath (~10 minutes).
The test solutions were prepared by the appropriate diluting of this stock solution and distributed into test vessels prior to introduction of algae.

Test organisms (species):

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

Details on test organisms:

Species: Pseudokirchneriella subcapitata (formerly known as Selenastrum capricornutum)
Strain number: 61.81 SAG (identical strains: CCAP 278/4; UTEX 1648; ATCC 22662)
Source: The algae were supplied by the SAG: Collection of Algal Cultures, Inst. Plant Physiology, and University of Göttingen, GERMANY. Cultured under standardised conditions (see OECD 201) in the Ecotoxicological Laboratory of CiToxLAB Hungary Ltd.
Justification of species: The species of Pseudokirchneriella subcapitata used, being a fast-growing species, is convenient for culturing and testing and is a recommended species by relevant guidelines.
Culture conditions: Stock cultures are small algal colonies that are inoculated onto agar regularly. These are transferred to fresh agar medium at least once every two months and are maintained under standardised conditions according to the test guidelines.
The pre-culture is intended to give a quantity of algae suitable for the inoculation of test cultures. The pre-culture was prepared with the OECD algal growth medium, incubated under the same conditions as the test and used when still growing exponentially, normally after an incubation period of about three days. When the algal cultures contain deformed or abnormal cells, they were discarded.

Test type:

not specified

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

No post exposure observation period specified in the study report.

Hardness:

Not specified

Test temperature:

Culture temperature was checked at the beginning of the experiment and each day thereafter in a flask filled with water, in the climatic chamber. In addition, water temperature was continuously measured (with a min/max thermometer) within the climate chamber. The temperature was in the range of 22.7 – 22.9 °C measured in the flask and between 22.5 and 23.2 °C measured within the climate chamber.

pH:

The pH was checked at the beginning and at the end of the test, in the control and each concentration. The pH of the control medium was not increased by more than 1.5 units during the test. The range of the pH was 7.31 – 9.29 during the experiment.

Dissolved oxygen:

Not specified

Salinity:

Not specified

Conductivity:

Not specified

Nominal and measured concentrations:

Because significant inhibition was observed at the highest examined concentration level during the preliminary range-finding test, five test concentrations in a geometric series with a separation factor of 3.2 and one control were tested in the main experiment.
The following nominal concentrations were tested: 1.0, 3.1, 9.8, 31.3 and 100.0 mg/L.
The corresponding measured geometric mean test item concentrations were: 0.28, 1.22, 3.88, 12.79 and 38.40 mg/L.
As the analytically measured concentration deviated more than 20 per cent from the nominal in most cases, the biological results are based on the measured geometric mean concentrations

Details on test conditions:

DESCRIPTION OF THE TEST PROCEDURE
The exposure time was 72 hours. The test was started (0 hours) by inoculation of a biomass of approximately 104 algal cells per mL test medium.
The test was performed with three replicates per test concentration and six replicates in the control group. Volumes of 100 mL algal suspension per replicate in 250 mL
Erlenmeyer flasks were continuously shaken by a laboratory orbital shaker to keep algae in suspension. The flasks were covered with air-permeable stoppers.

Preliminary Range Finding Test
A concentration range-finding test was conducted to determine the approximate toxicity of the test item so that appropriate test concentrations could be selected for use in the definitive test. Algal cells were exposed to each concentration of the test item plus a control, for 72 hours. The test was performed with two replicates per each test concentration and three replicates in the control group.

OBSERVATIONS
The cell numbers were determined at 24, 48 and 72 hours after starting the test by manual cell counting using a microscopic method with a counting chamber.
Microscopic observation of the algal cells in each concentration and in the control was performed (at 24h, 48h and 72h) to detect any abnormal appearance of the algae.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate is (Batch Number: A0345704)

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.22 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

other: growth rate, yield & biomass

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

3.88 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

other: Growth rate, yield and biomass

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

5.62 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

2.89 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

cell number

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

3.16 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

VALIDITY
The cell density in the control cultures increased by the factor of 70.33 within three days.
The mean coefficient of variation for section-by-section specific growth rates (days 0-1; 1-2; 2-3) in the control cultures was 10.87 %.
The coefficient of variation of average specific growth rates during the whole test period (day 0-3) in the control cultures was 0.62 %.
All validity criteria were met, therefore the study can be considered as valid.

CONCENTRATIONS OF THE TEST ITEM
Test concentrations were analytically determined at the start and of the experiment and 24 hour intervals thereafter during the test.
The following nominal concentrations were tested: 1.0, 3.1, 9.8, 31.3 and 100.0 mg/L.
The corresponding measured geometric mean test item concentrations were: 0.28, 1.22, 3.88, 12.79 and 38.40 mg/L.
Biological results are related to the geometric mean of the measured test item concentrations.

MORPHOLOGICAL DEVIATIONS OF THE ALGAL CELLS
Chuff cells were observed at the tested concentrations of 3.88 and 12.79 mg/L (measured), while thin cells were noted at the highest examined concentration of 38.40 mg/L (measured) during the test.

CELL NUMBERS
The cell number in each flask was determined at the 24th, 48th, 72nd hours.

AVERAGE SPECIFIC GROWTH RATES
The results of the statistical evaluation (based on Bonferroni t-Test; α=0.05) show that the 0-72 h average specific growth rate was statistically significantly different from the untreated control value at the concentration range 3.88 - 38.40 mg/L (measured), accordingly the No Observed Effect Concentration (NOEC) was determined as 1.22 mg/L (measured).
The 72 h ErC50 was determined [by Probit analysis (TOXSTAT software)] as 5.62 mg/L (95 % confidence limits: 4.92 – 6.42 mg/L).

AREAS UNDER THE GROWTH CURVES
The results of the statistical evaluation (based on Bonferroni t-Test; α=0.05) show that the 0-72 h areas were statistically significantly different from the untreated control value at the concentration range of 3.88 - 38.40 mg/L (measured), accordingly the No Observed Effect Concentration (NOEC) determined as 1.22 mg/L (measured).
The 72 h EbC50 value was determined [by Probit analysis (TOXSTAT software)] as 3.16 mg/L (95 % confidence limits: 2.79 – 3.58 mg/L).

YIELD
The results of the statistical evaluation (based on Bonferroni t-Test; α=0.05) show that the 0-72 h yield was statistically significantly different from the untreated control value at the tested concentration range of 3.88 - 38.40 mg/L (measured), accordingly the No Observed Effect Concentration (NOEC) determined as 1.22 mg/L (measured).
The 72 h EyC50 value was determined [by Probit analysis (TOXSTAT software)] as 2.89 mg/L (95 % confidence limits: 2.60 – 3.22 mg/L).

Results with reference substance (positive control):

For the evaluation of the quality of the algae and validation of the experimental conditions, Potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions.
The date of the last study (Study Code: 17/058-022AL) with the reference item Potassium dichromate is (Batch Number: A0345704): 07 – 10 March 2017.
The 72h ErC 50: 0.88 mg/L, (95 % confidence limits: 0.81 – 0.96 mg/L)
The 72h EyC 50: 0.54 mg/L, (95 % confidence limits: 0.50 – 0.59 mg/L)
The 72h EbC 50: 0.64 mg/L, (95 % confidence limits: 0.59 – 0.70 mg/L)

Reported statistics and error estimates:

The section-by-section specific growth rates in the control cultures were assessed (calculated as the specific growth rates for each day during the course of the test (days 0-1, 1-2 and 2-3) and to demonstrate exponential growth for the entire study period.
The inhibition of alga growth was determined from the biomass (area under the growth curves, A), the average specific growth rate (r) and from the yield (y). Mean values and standard deviations were calculated for each concentration at the start, and at the end of the test using Excel 2007 for Windows software (Microsoft Co./One Microsoft Way/Redmond, WA 98052-6399).
The ErC50, EbC50 and EyC50 values of the test item and their confidence limits were calculated using Probit analysis by TOXSTAT software.
Statistical comparisons of biomass, average specific growth rates and yield in controls and in the treated groups were carried out using analysis of variance (ANOVA) and Bonferroni t-Test (α = 0.05) by TOXSTAT software.
For the determination of the LOEC and NOEC, the calculated mean biomass, growth rates and yield at the test concentrations were tested on significant differences to the control values by Bonferroni t-Test.

Results of the Preliminary Range-Finding Test

Nominal concentrations [mg/L]	Untreated control	0.1	1	10	100
Average of cell number at 72 hours (x 10^4cell/mL)	79.67	76.50	74.50	12.50	1.00

 

Calculation of exposure concentrations

Nominal concentration (mg/L)	Measured concentrations (mg/L)				Geometric mean
	0h/start	24h	48h	72h/end	mg/L
Control	n.d.	n.d.	n.d.	n.d.	-
1.0	0.993	0.525	0.241	0.050*	0.28
3.1	3.10	1.70	0.865	0.488	1.22
9.8	9.67	5.30	2.76	1.61	3.88
31.3	30.6	17.1	9.19	5.57	12.79
100.0	95.7	51.9	26.7	16.4	34.40

n.d. – not detected

*Remark: Concentration was below the Limit of Quantification. In order to calculate mean value, concentration was taken as the half of the Limit of Quantification (LOQ = 0.10 mg/L) according to OECD 23; paragraph 3.3

 

Growth Rates (μ) and Percentage Inhibition of μ during the Test Period

Concentration		Growth rate (μ) and % inhibition of μ
Nominal [mg/L]	Measured [mg/L]	0-24 h		0 – 48 h		0 – 72 h
		μ	%	μ	%	μ	%
Control	0.0	0.0589	0.0	0.0598	0.0	0.0591	0.0
1.0	0.28	0.0578	1.9	0.0598	-0.1	0.0591	0.0
3.1	1.22	0.0569	3.4	0.0589	1.4	0.0590	0.1
9.8	3.88	0.0458	22.2	0.0395*+	34.0	0.0381*	35.5
31.3	12.79	0.0193*	67.3	0.0144*	75.8	0.0096*	83.7
100.0	38.40	0.0000*	100.0	0.0000*	100.0	0.0000*	100.0

*: statistically significant different compared to the control values (Bonferroni t-Test; α = 0.05)

+: at these values the rounding of the EXCEL and TOXSTAT software was different. The table contains the values calculated with EXCEL.

 

Area under Growth Curves (A) and Percentage Inhibition of A during the Test Period

Concentration		Area under the Growth Curves (A) and % inhibition of A
Nominal [mg/L]	Measured [mg/L]	0-24 h		0 – 48 h		0 – 72 h
		μ	%	μ	%	μ	%
Control	0.0	38.0	0.0	276.0	0.0	1308.0	0.0
1.0	0.28	36.0	5.3	272.0	1.4	1304.0	0.3
3.1	1.22	36.0	5.3	264.0	4.3	1284.0	1.8
9.8	3.88	24.0	36.8	116.0*	58.0	360.0*	72.5
31.3	12.79	8.0*	78.9	28.0*	89.9	52.0*	96.0
100.0	38.40	0.0*	100.0	0.0*	100.0	0.0*	100.0

*: statistically significant different compared to the control values (Bonferroni t-Test; α = 0.05)

 

Yield (Y) and Percentage Inhibition of Y during the Test Period

Concentration		Concentration
Nominal [mg/L]	Measured [mg/L]	0-72 h
		Y	%
Control	0.0	69.3	0.0
1.0	0.28	69.3	0.0
3.1	1.22	69.0	0.5
9.8	3.88	14.7*	78.8
31.3	12.79	1.0*	98.6
100.0	38.40	0.0*	100.0

*: statistically significant different compared to the control values (Bonferroni t-Test; α = 0.05)

 

pH-Values in the Test Media at the Start and End of the Test

Concentration		pH-Values
Nominal [mg/L]	Measured [mg/L]	Start	End
Control	0.0	7.86	9.27 9.22 9.21 9.25 9.27 9.29
1.0	0.28	7.88	8.91 8.94 8.97
3.1	1.22	7.85	8.80 8.81 8.79
9.8	3.88	7.81	7.80 7.83 7.77
31.3	12.79	7.79	7.75 7.73 7.78
100.0	38.40	7.65	7.37 7.32 7.31

 

Temperature in the Climate Chamber and in the Test Media during the Test

Parameter		Exposure Time
		Day 0	Day 1	Day 2	Day 3
Temperature (°C) measured in flask		22.9	22.8	22.7	22.8
Min / max temperature (°C) of the climate chamber	Max	23.1	22.9	23.2	23.0
	Min	22.9	22.6	22.5	22.7

 

Cell Number (x 10⁴cell/mL) determined in the Main Experiment

Concentration			Number of cells
Nominal [mg/L]	Measured [mg/L]		0 h	24 h	48 h	72 h
Control	0.0	Mean SD	1 1 1 1 1 1 1.00 0.0	5 5 4 4 4 3 4.17 0.8	17 18 17 19 16 19 17.67 1.2	70 72 69 73 68 70 70.33 1.9
1.0	0.28	Mean SD	1 1 1 1.00 0.0	4 4 4 4.00 0.0	17 18 18 17.67 0.6	69 73 69 70.33 2.3
3.1	1.22	Mean SD	1 1 1 1.00 0.0	3 5 4 4.00 1.0	18 15 18 17.00 1.7	70 67 73 70.00 3.0
9.8	3.88	Mean SD	1 1 1 1.00 0.0	3* 3* 3* 3.00 0.0	7* 7* 6* 6.67 0.6	17* 13* 17* 15.67 2.3
31.3	12.79	Mean SD	1 1 1 1.00 0.0	1* 2* 2* 1.67 0.6	2* 2* 2* 1.00 0.0	2* 2* 2* 2.00 0.0
100.0	38.40	Mean SD	1 1 1 1.00 0.0	1+ 1+ 1+ 1.00 0.0	1+ 1+ 1+ 1.00 0.0	1+ 1+ 1+ 1.00 0.0

*: Chuff, deformed cells

+: Thin, deformed cells

Validity criteria fulfilled:

yes

Conclusions:

The effect of PTSM test item was assessed on algal growth using the unicellular green alga Pseudokirchneriella subcapitata (formerly known as Selenastrum capricornutum), over an exposure period of 72 hours.
With respect to the inhibitory effect of the test item, the 0-72 h average specific growth rates, areas and yield were significantly different from that of the control group in the examined concentration range of 3.88 – 38.40 mg/L (measured), therefore the NOEC was determined as 1.22 mg/L (measured) and LOEC was determined as 3.88 mg/L (measured).

Executive summary:

The effect of test item was assessed on algal growth using the unicellular green alga Pseudokirchneriella subcapitata (Selenastrum capricornutum), over an exposure period of 72 hours.

 

As significant inhibition was observed at the highest examined concentration level during the preliminary range-finding test, five test concentrations in a geometric series (factor 3.2) and one control were tested in the main experiment.

 

The nominal concentrations of test item used in the main experiment were: 1.0, 3.1, 9.8, 31.3 and 100.0 mg/L.

 

Test concentrations were analytically determined at the start and of the experiment and 24 hour intervals thereafter during the test. The corresponding measured geometric mean test item concentrations were: 0.28, 1.22, 3.88, 12.79 and 38.40 mg/L.

 

As the analytically measured concentration deviated more than 20 per cent from the nominal in most cases, the biological results are based on the measured geometric mean concentrations.

 

The test design included three replicates at each test concentration and six replicates for the untreated controls.

 

Statistical comparisons of biomass, average specific growth rates and yield in control and in treated groups were carried out using analysis of variance (ANOVA) and Bonferroni t-Test (α = 0.05) by TOXSTAT software.

The ErC50, EbC50 and EyC50 values of the test item and their confidence limits were calculated using Probit analysis by TOXSTAT software.

 

With respect to the inhibitory effect of the test item, the 0-72 h average specific growth rates, areas and yield were significantly different from that of the control group in the examined concentration range of 3.88 – 38.40 mg/L (measured), therefore the NOEC was determined as 1.22 mg/L (measured) and LOEC was determined as 3.88 mg/L (measured). The biological results are summarised in the following table:

 

Influence of PTSM on the Growth of Pseudokirchneriella subcapitata

Parameter (0-72 h)	Growth rate (r) [mg/L]	Yield (y) [mg/L]	Biomass (b) [mg/L]
Calculation based on measured geometric mean concentrations
EC50 95% conf. limits	5.62 4.92 – 6.42	2.89 2.60 – 3.22	3.16 2.79 – 3.58
NOEC LOEC	1.22 3.88	1.22 3.88	1.22 3.88

 

Description of key information

the NOEC was determined as 1.22 mg/L (measured) and LOEC was determined as 3.88 mg/L (measured).

Key value for chemical safety assessment

EC50 for freshwater algae:

2.89 mg/L

EC10 or NOEC for freshwater algae:

1.22 mg/L

Additional information

The effect of test item was assessed on algal growth using the unicellular green alga Pseudokirchneriella subcapitata (Selenastrum capricornutum), over an exposure period of 72 hours.

 

As significant inhibition was observed at the highest examined concentration level during the preliminary range-finding test, five test concentrations in a geometric series (factor 3.2) and one control were tested in the main experiment.

 

The nominal concentrations of test item used in the main experiment were: 1.0, 3.1, 9.8, 31.3 and 100.0 mg/L.

 

Test concentrations were analytically determined at the start and of the experiment and 24 hour intervals thereafter during the test. The corresponding measured geometric mean test item concentrations were: 0.28, 1.22, 3.88, 12.79 and 38.40 mg/L.

 

As the analytically measured concentration deviated more than 20 per cent from the nominal in most cases, the biological results are based on the measured geometric mean concentrations.

 

The test design included three replicates at each test concentration and six replicates for the untreated controls.

 

With respect to the inhibitory effect of the test item, the 0-72 h average specific growth rates, areas and yield were significantly different from that of the control group in the examined concentration range of 3.88 – 38.40 mg/L (measured), therefore the NOEC was determined as 1.22 mg/L (measured) and LOEC was determined as 3.88 mg/L (measured). The biological results are summarised in the following table:

 

Influence of PTSM on the Growth of Pseudokirchneriella subcapitata

Parameter (0-72 h)	Growth rate (r) [mg/L]	Yield (y) [mg/L]	Biomass (b) [mg/L]
Calculation based on measured geometric mean concentrations
EC50 95% conf. limits	5.62 4.92 – 6.42	2.89 2.60 – 3.22	3.16 2.79 – 3.58
NOEC LOEC	1.22 3.88	1.22 3.88	1.22 3.88