Butyl 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:

20 January 2020 to 30 January 2020

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:

2011

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OCSPP 850.4500 (Algal Toxicity)

Version / remarks:

2012

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Concentrations were adjusted for the purity of the test material.

Analytical monitoring:

yes

Details on sampling:

Exposure solutions were sampled at 0 hours prior to division into replicate vessels. At 96 hours samples were taken from a composite of all replicates within each treatment level and control; additional replicate for solution without algae. Samples removed at 96 hours were centrifuged prior to analysis (in Teflon centrifuge tubes at 3000 rpm for 10 minutes) to remove algal biomass.
One sample was taken at each interval. Additional samples of the exposure solutions were also collected at each sampling interval and stored frozen as archive samples.
Samples were taken from the approximate midpoint from the surface, bottom, and sides of the vessel with a pipet.
Three quality control samples were taken at at 0 hours and six at 96 hours, prepared in AAP medium at nominal concentrations bracketing the concentration tested. Three of the six QC samples at 96 hours were centrifuged prior to analysis to evaluate the loss of test substance due to centrifugation.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
The primary stock solution was prepared by adding the test material to a 4.0 L clear, glass aspirator bottle with a bottom side drain, containing 2.0 L of AAP medium. The solution was set to slowly mix overnight using a magnetic stir plate and Teflon-coated stir bar. The solution was observed to be clear and colourless with visible, oily globules on the bottom of the vessel as well as on the surface of the solution. Following mixing and a 5-minute settling period, the solution was observed to be clear and colourless with visible, white, oily globules on the bottom of the vessel and clear, oily globules on the surface of the solution. Approximately 1.5 L of the soluble portion of the solution was drained from the aspirator bottle, while avoiding the bottom and surface of the solution. The resulting solution was clear and colourless with no visible undissolved test material and was used to prepare exposure solutions, by further dilution with AAP medium.
The resulting exposure solutions were observed to be clear and colourless with no visible undissolved test material following mixing and inversion of the volumetric flasks.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: 1648
- Source: UTEX The Culture Collection of Algae at the University of Texas, Austin, Texas (maintained in stock culture at the Test Facility)
- Age of inoculum (at test initiation): three days since previous transfer
- Culturing media and conditions: same as test

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Test temperature:

22 - 23 °C

pH:

6.8 - 9.7

Conductivity:

99 - 110 µS/cm

Nominal and measured concentrations:

Nominal Loading Rates: 1.0, 2.6, 6.4, 16, 40, and 100% of a 100 mg/L water-accommodated fraction (WAF)
Geometric Mean Measured Concentrations: 0.17, 0.45, 1.1, 2.7, 7.1, and 19 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 250-mL glass flasks, fitted with stainless steel caps which permitted gas exchange
- Fill volume: 100 mL per replicate
- Aeration: continuous agitation at a rate of 100 rpm
- Initial cells density: 10,000 cells/mL
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 8
- Additional replicates: Replicate flasks (E and F) of the 6.4% of a 100 mg/L WAF (nominal) treatment level, not inoculated with algae, analysed at 96 hours of exposure. Results of the solution without algae were compared with the results for the 6.4% of a 100 mg/L WAF solution containing algae to estimate the impact that the presence of algal biomass had on the test material concentration.

GROWTH MEDIUM
- Standard medium used: yes (AAP (Algal Assay Procedure) medium prepared with sterile, deionized source water)
- Detailed composition: The composition of Algal Assay Procedure (AAP) Medium was as follows: NaNO3 (25.5 mg/L), MgCl2.6H2O (12.16 mg/L), CaCl2.2H2O (4.41 mg/L), MgSO4.7H2O (14.7 mg/L), K2HPO4.3H2O (1.368 mg/L), NaHCO3 (15.0 mg/L), H3BO3 (185.5 µg/L), Na2SeO4a (1.88 µg/L), MnCl2.4H2O (415.4 µg/L), ZnCl2 (3.270 µg/L), CoCl2.6H2O (1.43 µg/L), CuCl2.2H2O (0.012 µg/L), Na2MoO4.2H2O (7.26 µg/L), FeCl3.6H2O (160 µg/L), Na2EDTA.2H2O (300 µg/L)

TEST MEDIUM / WATER PARAMETERS
- Test medium: AAP (Algal Assay Procedure) medium prepared with sterile, deionized source water
- Dilution Water Source (Culture and Testing): Town of Wareham water
- Total Organic Carbon: 0.65 mg/L
- Culture medium different from test medium: no
- Intervals of water quality measurement: Representative samples of the dilution water source used in the preparation of the culture medium were analysed periodically for the presence of pesticides, PCBs, and toxic metals

OTHER TEST CONDITIONS
- Adjustment of pH: initial pH adjusted with dilute hydrochloric acid (HCl) or sodium hydroxide (NaOH), to 7.5 ± 0.1, prior to use if necessary
- Photoperiod: None (continuous) Photosynthically-Active Radiation (62 to 83 µE/m²/S)
- Light intensity: 5500 to 7000 lux

EFFECT PARAMETERS MEASURED
- Algal Growth: At each 24-hour interval, cell counts were conducted on each replicate solution of the treatment levels and the control using a Beckman Coulter Multisizer 4e Particle Analyser. Generally, 2 samples were removed from each test vessel, and two counts were determined for each sample. The 2 counts determined for each sample were averaged, and the 2 average cell counts determined for each replicate were averaged. The reported cell density was the average of the 4 cell counts determined for each replicate. Observations of the health of the algal cells were also made and recorded at each 24 hour interval. These observations were made in an alternating replicate of each control or treatment group using a hemacytometer (Neubauer Improved) and compound microscope.
- Solution for Algistatic/Algicidal Properties: At exposure termination, a composite solution was prepared from the four 40% of a 100 mg/L WAF (nominal) replicate exposure vessels. From this composite, a sample was removed and then diluted with freshly prepared AAP medium to prepare a subculture with a nominal loading rate of 1.0% of a 100 mg/L WAF, equivalent to the lowest nominal test concentration. The subculture was incubated for up to 9 days under conditions consistent with those maintained during the definitive exposure. During this period, the subculture was examined microscopically every other day to determine whether or not cell growth had resumed. The subculture was discontinued after a substantial increase in cell density (i.e., > 10x) was observed. If an increase in cell density was not observed after 9 days, the vessel was terminated.
- Physicochemical properties of water: pH, conductivity and temperatures measurements were taken at exposure initiation on the exposure solutions remaining in the mixing flasks after the individual exposure flasks had been filled. At exposure termination, after cell counts were completed, samples were removed from the replicate vessels of each treatment level and the control and were respectively composited for pH measurement.

CALCULATION
Growth rate was calculated from the daily cell density counts; specific growth rate between inoculation density and the cell density of the specified time interval calculated using the following equation:
specific growth rate (µ) = (ln Xn - ln Xn0) / (tn - tn0)
where
Xn = cell density at the beginning of the specified time interval tn (days)
Xn0 = cell density at the previous time interval (e.g., tn0) in cells/mL
tn = time at observation interval n in days, based on actual time
tn0 = time at the previous observation interval (e.g., exposure initiation) in days, and based on actual time elapsed between observation intervals

TEST CONCENTRATIONS
A preliminary test utilised two exposure vessels for each of the following loading rates: 0 (control), 0.10, 1.0, 10, and 100 % of a 100 mg/L WAF. Findings on the percent inhibition at 96 hours (in terms of cell density), were used to establish the nominal loading rates for the 96-hour definitive exposure.

Reference substance (positive control):

yes

Remarks:

Zinc chloride (ZnCl2)

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

39 other: % of a 100 mg/L WAF

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% Confidence Intervals: 38 - 40 % of a 100 mg/L WAF

Key result

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

7 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% Confidence Intervals: 6.7 - 7.2 mg/L

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

6.4 other: % of a 100 mg/L WAF

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

1.1 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

ANALYTICAL RESULTS
Measured concentrations declined throughout the exposure and maintained the expected concentration gradient.
The analytical result of the 96-hour samples from the 6.4% of a 100 mg/L WAF nominal loading rate solution, with algae present, was 0.50 mg/L. The equivalent test solution without algae present resulted in a recovery of 0.55 mg/L, and demonstrated that the presence of algae had no impact on the concentration of test material in the exposure solution.
Analysis of the QC samples resulted in measured concentrations which were consistent with the predetermined recovery range and ranged from 108 to 119% (N = 9) of the nominal fortified concentrations (0.0500 to 21.4 mg/L). Based on these results, it was determined that the appropriate accuracy and quality control were maintained during the analysis of the exposure solutions. The comparison of the results for the centrifuged and uncentrifuged samples indicates that centrifugation of the test samples had no impact on the measured concentrations.

BIOLOGICAL RESULTS
During the 96-hour exposure, cells observed in the control and all the treatment levels tested were observed to be healthy and normal in appearance, with the exception of the 40 and 100% of a 100 mg/L WAF treatments where thin cell walls were observed at 72 and 96 hours of exposure. Additionally, at 96 hours of exposure the cells in the 100% of a 100 mg/L WAF were observed to be fragmented under microscopic evaluation. As a Beckman Coulter Multisizer 4e Particle AnalySer was used for this evaluation, the fragmentation artificially inflated the cell density counts at this interval as it was counting each fragmented cell portion as an individual cell. Fragmentation was confirmed by comparing the particle size of the control and the high treatment level particle counter data for this interval. The mean particle size for the controls and the high treatment level were 4.126 and 2.878 um, respectively. For this reason, the data for the high loading rate wa not included in the statistical analysis. At each observation interval, control and exposure solutions were observed to be the same as at exposure initiation with respect to precipitation, clarity, and material adhered to the sides of the vessels.

ALGISTATIC/ ALGICIDAL RECOVERY PERIOD
The subculture prepared from the 40% of a 100 mg/L WAF solution for the algistatic/algicidal determination had a test material concentration of 1.0% of a 100 mg/L WAF, the lowest nominal loading rate tested, and an estimated cell density of 3343 cells/mL. The subculture was agitated continuously at a rate of 100 ± 10 rpm on an orbital shaker and was maintained at a temperature range of 22 to 24 °C and a light intensity range of 72 to 78 µE/m²/S. After 6 days, a cell density of 425,400 cells/mL was measured in the subculture, corresponding to an approximately 127-fold increase in cell density over 6 days. This observation indicates that the test material has an algistatic, rather than algicidal effect on the growth of R. subcapitata at a nominal concentration of 40% of a 100 mg/L WAF.

Results with reference substance (positive control):

A reference test conducted at the Test Facility outside the scope of this study was used to establish the reliability of the exposure conditions and evaluate the health and sensitivity of Raphidocelis subcapitata. Under the conditions of the study the 96-hour EC50 was determined to be 0.11 mg Zn/L, with a 95% confidence interval of 0.066 to 0.16 mg Zn/L (historical mean = 0.087 mg Zn/L, March 2005 to present).
The result was within the expected range for Raphidocelis subcapitata; therefore, the exposure conditions are considered reliable and the culture is considered of appropriate health and sensitivity for use in toxicity testing.

Reported statistics and error estimates:

Prior to NOEC and LOEC determinations, the data were first checked for normality using Shapiro-Wilk’s Test and for homogeneity of variance using Bartlett’s Test If the data sets passed the tests for homogeneity and normality, then a parametric statistical test, e.g., Williams’ Multiple Comparison Test was used to determine the NOEC and LOEC. If the data did not pass the tests for homogeneity and normality, then the NOEC and LOEC were determined using an appropriate non-parametric statistical test, e.g., Jonckheere-Terpstra’s Step-Down Test. All statistical determinations were made at the 95% level of certainty, except in the case of Shapiro-Wilk’s and Bartlett’s Tests, where the 99% level of certainty was applied.
EC10, EC20, and EC50 values were calculated using a nonlinear regression model. CETIS Version 1.9 was used to perform all statistical calculations.

Concentrations Measured in the Exposure Solutions

Nominal Loading Rate (% of a 100 mg/L WAF)	Measured Concentration (mg/L)			Geometric Mean^a
	0 Hour	96 Hour	Percent Decline from 0 Hour^a
Control	<0.13^b	<0.044	NA^c	NA
1.0	0.37	0.078	79	0.17
2.6	0.95	0.21	78	0.45
6.4	2.5	0.50/0.55^d	80	1.1
16	5.9	1.3	78	2.7
40	15	3.4	77	7.1
100	36	9.7	73	19

a Geometric mean measured concentration and percent decline values were calculated using actual analytical data and not the rounded values presented in this table.

b Concentrations expressed as less than values were below the method detection limit (MDL).  The MDL is dependent upon the lowest concentration calibration standard and the dilution factor of the controls.

c NA = Not Applicable

d Result of the additional sample without algae present to determine biological uptake/degradation

Calcultaed Growth Rates

Nominal Loading Rate (% of a 100 mg/L WAF)	Geometric Mean Measured Concentration (mg/L)	Replicate	Growth Rate (day^-1)
			Observation Interval (Hours)
			0 - 24 (0.941 days)	0 - 48 (1.96 days)	0 - 72 (2.91 days)	72-Hour % Inhibition^ab	0 - 96 (3.93 days)	96-Hour % Inhibition^ab	96-Hour % CV^b
Control	Control	A	1.64	1.51	1.45		1.36
		B	1.56	1.60	1.50		1.39
		C	1.30	1.36	1.36		1.30
		D	1.50	1.56	1.44		1.36
		E	1.40	1.47	1.40		1.32
		F	1.60	1.61	1.45		1.36
		G	1.61	1.55	1.42		1.35
		H	1.62	1.46	1.42		1.36
		Mean (SD)^b	1.53 (0.12)	1.52 (0.08)	1.43 (0.04)	NA^c	1.35 (0.03)	NA	2.1
1.0	0.17	A	1.41	1.48	1.42		1.36
		B	1.29	1.46	1.42		1.35
		C	1.50	1.49	1.41		1.37
		D	1.66	1.55	1.49		1.37
		Mean (SD)	1.47 (0.15)	1.49 (0.04)	1.43 (0.04)	0	1.36 (0.01)	-1	0.9
2.6	0.45	A	1.31	1.46	1.36		1.30
		B	1.40	1.47	1.44		1.34
		C	1.39	1.37	1.37		1.34
		D	1.26	1.45	1.45		1.36
		Mean (SD)	1.34 (0.07)	1.44 (0.05)	1.41 (0.05)	2	1.34 (0.02)	1	1.8
6.4	1.1	A	1.33	1.37	1.37		1.29
		B	1.53	1.49	1.39		1.34
		C	1.60	1.42	1.42		1.36
		D	1.35	1.47	1.42		1.32
		Mean (SD)	1.45 (0.13)	1.44 (0.05)	1.40 (0.02)	2	1.33 (0.03)	2	2.1
16	2.7	A	1.15	1.21	1.25		1.19
		B	1.10	1.25	1.26		1.26
		C	1.24	1.22	1.27		1.25
		D	1.21	1.21	1.25		1.19
		Mean (SD)	1.17 (0.06)	1.22 (0.02)	1.26^d(0.01)	12	1.23^e(0.04)	9	2.9
40	7.1	A	0.58	0.50	0.84		0.62
		B	0.62	0.43	0.78		0.73
		C	0.47	0.43	0.68		0.66
		D	0.54	0.39	0.73		0.62
		Mean (SD)	0.55 (0.07)	0.44 (0.05)	0.76^d(0.07)	47	0.66^e(0.05)	51	8.0
100	19	A	0.44	0.41	0.79		0.96
		B	0.40	0.24	0.65		0.96
		C	0.39	0.30	0.94		1.01
		D	0.32	0.35	0.87		1.00
		Mean (SD)	0.39 (0.05)	0.33 (0.07)	0.81 (0.13)	43	0.98 (0.03)	27	3.0

a Percent inhibition relative to the control.

b Mean, standard deviation (SD), percent inhibition, and % CV are calculated from original raw data, not from the rounded values present in this table.

c NA = Not Applicable

d Significantly reduced compared to the control, based on Jonckheere-Terpstra’s Step-Down Test.

e Significantly reduced compared to the control, based on Williams’ Multiple Comparison Test.

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the study, the 96-hour ErC50 was determined to be 39% of a 100 mg/L WAF (7.0 mg/L meas., geo. mean), while the 96-hour NOEC was determined to be 6.4% of a 100 mg/L WAF (1.1 mg/L meas., geo. mean).

Executive summary:

The effect of the test material on the growth of the freshwater green algae, Raphidocelis subcapitata was investigated in a study which was conducted in accordance with the standardised guidelines OECD 201 and OCSPP 850.4500, and under GLP conditions.

During the study the Raphidocelis subcapitata was exposed to water accomodated fractions of test material at nominal loading rates of 1.0, 2.6, 6.4, 16, 40, and 100% of a 100 mg/L water-accommodated fraction (WAF) under static test conditions. Analysis of the test solutions by gas chromatography revealed that these nominal loading rates related to geometric mean measured concentrations of 0.17, 0.45, 1.1, 2.7, 7.1, and 19 mg/L. Following 96 hours exposure, cells counts were determined and used for the calculation of the specfic growth rates.

Under the conditions of the study, the 96-hour ErC50 was determined to be 39% of a 100 mg/L WAF (7.0 mg/L meas., geo. mean), while the 96-hour NOEC was determined to be 6.4% of a 100 mg/L WAF (1.1 mg/L meas., geo. mean).

Description of key information

The 96-hour ErC50 was determined to be 39% of a 100 mg/L WAF (7.0 mg/L meas., geo. mean), while the 96-hour NOEC was determined to be 6.4% of a 100 mg/L WAF (1.1 mg/L meas., geo. mean).

Key value for chemical safety assessment

EC50 for freshwater algae:

7 mg/L

EC10 or NOEC for freshwater algae:

1.1 mg/L

Additional information

The effect of the test material on the growth of the freshwater green algae, Raphidocelis subcapitata was investigated in a study which was conducted in accordance with the standardised guidelines OECD 201 and OCSPP 850.4500, and under GLP conditions.

During the study the Raphidocelis subcapitata was exposed to water accomodated fractions of test material at nominal loading rates of 1.0, 2.6, 6.4, 16, 40, and 100% of a 100 mg/L water-accommodated fraction (WAF) under static test conditions. Analysis of the test solutions by gas chromatography revealed that these nominal loading rates related to geometric mean measured concentrations of 0.17, 0.45, 1.1, 2.7, 7.1, and 19 mg/L. Following 96 hours exposure, cells counts were determined and used for the calculation of the specfic growth rates.

Under the conditions of the study, the 96-hour ErC50 was determined to be 39% of a 100 mg/L WAF (7.0 mg/L meas., geo. mean), while the 96-hour NOEC was determined to be 6.4% of a 100 mg/L WAF (1.1 mg/L meas., geo. mean).