Benzene, mono-C10-13-alkyl derivs., distn. residues, sulfonated, barium salts

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP guideline study. Responsibility of the study data lies on the master dossier for the main read-across substance. The original study was not handed out.

Qualifier:

according to guideline

Guideline:

EPA OTS 797.1050 (Algal Toxicity, Tiers I and II)

Deviations:

yes

Remarks:

The determination of algistatic vs algicidal effects was omitted as the study was completed as a limit test

GLP compliance:

yes

Remarks:

No details available.

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material:
The test substance is chemically similar to the submission substance being described as alkaryl sulphonate.
- Name of material: Benzenesulfonic acid, C14-24 branched/linear alkyl derivative, calcium salt
- CAS: 115733-09-0

Analytical monitoring:

yes

Details on sampling:

The number of algal cells/mL in each test vessel and the occurrence of relative size differences, unusual cell shapes, color, flocculation, adherence of cells to test containers, or aggregation of cells was determined visually by means of direct microscopic examination with a hemocytometer. Cell counts were made and recorded daily during the 96 hour test.
Total organic carbon (TOC) measurements of initial (0 hours) high (1000 mg/L), low (100 mg/L) and control test solutions and at test termination (96 hours), conducted on each replicate control and treatment vessel.

Vehicle:

no

Details on test solutions:

Each of the three WAFs (100, 300, 1,000 mg/L) was individually prepared by combining the appropriate amount of test substance and dilution water in a glass mixing vessel equipped with a magnetic stirrer and stirring these mixtures for 24 hours, settling the mixtures for 1 hour, and siphoning the water phase containing the WAF. During the mixing of the WAF the vortex extended approximately 30 % of the distance from the surface to the bottom of the mixing vessel.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Green algae
- Source: Cells taken from a log-growth phase in-house culture originally purchased from University of Texas at Austin alga collection

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Remarks on exposure duration:

72-h readings were also performed.

Post exposure observation period:

No post exposure observation period described.

Hardness:

No details available.

Test temperature:

23.3 - 24 °C

pH:

Start of the test: 7.5 - 9.8
After 96 h: 8.3 - 10.1

Dissolved oxygen:

No details available.

Salinity:

Not applicable.

Nominal and measured concentrations:

Control, 100, 300 and 1000 mg/L WAF.
TOC levels were 2 - 8 mg/L in the control, 2 - 4 mg/L at 100 mg/L WAF, 2 - 3 mg/L at 300 mg/L WAF and 2 - 3 mg/L at 1000 mg/L WAF loading. TOC levels were not considered to be indicative of actual test material concentrations and results are therefore based on nominal loading rates.

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks containing 100 mL stoppered with foam plugs to reduce the entry of dust randomly placed on an orbital shaker to approximately 100 cycles per minute
- Renewal rate of test solution: Solution was not renewed.
- Initial cells density: 10000 cells/mL
- No. of vessels per concentration: 3
- Other test conditions: Test vessels were randomly arranged on a rotary shaker adjusted to 100 rpm in an incubator during the test (a random numbers table was used to select the location of each vessel).

GROWTH MEDIUM: Sterile enriched alga media adjusted to pH 7.5. Measured TOC and total suspended solids in fresh untreated alga media were < 1.0 and < 10 mg/L, respectively.

OTHER TEST CONDITIONS
- Sterile test conditions: Yes
- Adjustment of pH: Adjusted to pH 7.5
- Photoperiod: Constant light (24 hours per day)
- Light intensity: 47 - 50 µEin/m² x second

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: Visual determination by means of direct microscope examination with a hemocytometer

TEST CONCENTRATIONS
- Test concentrations: Control, 100, 300 and 1000 mg/L WAF

Reference substance (positive control):

no

Remarks:

Not mandatory under the applied guideline.

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

other: WAF loading rates

Basis for effect:

growth rate

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

other: WAF loading rates

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

other: WAF loading rates

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

other: WAF loading rates

Basis for effect:

biomass

Duration:

96 h

Dose descriptor:

EL50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

other: WAF loading rates

Basis for effect:

growth rate

Duration:

96 h

Dose descriptor:

EL50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

other: WAF loading rates

Basis for effect:

biomass

Details on results:

The control response was satisfactory.
At 72-hours, biomass measurements were 80 %, 62 % and 70 % of the control at 100, 300 and 1000 mg/L WAF, respectively. At 96-hours, biomass measurements were 70 %, 66 % and 88 % of the control at 100, 300 and 1000 mg/L WAF, respectively.

Results with reference substance (positive control):

Not applicable.

Reported statistics and error estimates:

A parametric one-way analysis of variance (ANOVA) and Dunnett's test were used to calculate the No Observed Effect Level (NOEL).

The test vessels containing 1000mg/L WAFs were slightly cloudy at the start of the test and at 24hours. The algal population grew well during the test, resulting in an average of 1711000 cells/mL in the control (doubling time: 13 hours). Cell growth data from the acute toxicity test with the WAF of 3 concentrations of substance and the freshwater alga, Selenastrum capricornutum are provided below with % growth rates compared to control are shown below at time points 0, 24, 48, 72 and 96 hours.

Table 1. Cell growth data

0 (control)		1	10	18	123	826	1,555
		2	10	21	145	1,008	1,866
		Mean	10	20	134	917	1,711
	%	Control	—	—	—	—	-—
100 mg/L WAF		1	10	15	118	680	1,250
		2	10	18	133	790	1,145
		Mean	10	17	126	735	1,198
	%	Control	100	85	94	80	70
300 mg/L WAF		1	10	<10	120	506	1,125
		2	10	<10	107	638	1,120
		Mean	10	<10	114	572	1,122
	%	Control	100	< 50	85	62	66
1000 mg/L WAF		1	10	<10	132	654	1,610
		2	10	<10	125	632	1,385
		Mean	10	<10	129	643	1,498
	%	Control	100	< 50	96	70	88

 

Based on the number of cells/mL or the average specific growth rate, the 72 and 96 hour EC50s are > 1000 mg/L.

No effects (size differences, unusual cell shapes, colours, flocculations, adherence of cells to test containers, or aggregation of cells) were noted. Based on the number of cells/mL or the average specific growth rate, the 72 and 96 hour NOEC values are 1000 mg/L.

Validity criteria fulfilled:

yes

Remarks:

Scientifically validity criteria were fulfilled.

Conclusions:

The report summarises a valid guideline study with GLP compliance. The test substance was not toxic to Selenastrum capricornutum at a loading rate up to 1000 mg/L nominal WAF, therefore the ErL50(96h) as well as the EbL50(96h) are considered to be > 1000mg/L.

Executive summary:

In a 96-hour acute toxicity study, the cultures of Selenastrum capricornutum (new name: Pseudokirchneriella subcapitata) were exposed to Benzenesulfonic acid, C14-24 branched/linear alkyl derivative, calcium salt (CAS 115733-09-0) at nominal concentrations of 100, 300 and 1000 mg/L as WAF loading rates under static conditions in accordance with the EPA Guideline OTS 797.1050 (Ward, 1994). Water Accommodated Fractions (WAFs) were prepared due to the intrinsic properties of the test substance (i.e. low water solubility). Daily cell counts and cell observations were made visually by means of direct microscopic examination with a hemocytometer. The algal population grew well during the test, since an average of 1,711,000 cells/mL were recorded in the control group; resulting in a doubling time of 13 h. The test vessels containing 1000 mg/L WAF were slightly cloudy at the start of the test and after 24 h exposure time. After 72 hours, biomass measurements were 80 %, 62 % and 70 % of the control at 100, 300 and 1000 mg/L WAF, respectively. At 96 hours, biomass measurements were 70 %, 66 % and 88 % of the control at 100, 300 and 1000 mg/L WAF, respectively. Based on these findings, the ErL50(72h), ErL50(96h), EbL50(72h) as well as the EbL50(96h) value is considered to be > 1000 mg/L. A parametric one-way analysis of variance (ANOVA) and Dunnett´s test were used to calculate the corresponding No Observed Effect Level (NOEL), which is reported as 1000 mg/L WAF for each sampling period.

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study meets criteria for Klimisch score 1 because it follows an internationally recognised method and was carried out according to Good Laboratory Practice.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

No further information is available.

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material:
- Physical state: Dark brown slightly viscous liquid
- Storage conditions: In the dark at room temperature

Analytical monitoring:

no

Details on sampling:

- Concentrations: 0 and 100 mg/L nominal WAF loading rate

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: 250 mg of test material was added to the surface of 2.5 litres of culture medium to give a 100 mg/L loading rate. The test material was stirred by a magnetic stirrer using a stirring rate such that a vortex was formed to give a slight dimple at the water surface. Stirring was conducted for 24 hours. The mixture was then allowed to stand for 1 hour. A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. The aqueous phase or WAF was removed by mid-depth siphoning (the first 75-100 mL discarded) to give the 100 mg/L loading rate WAF.
- Evidence of undissolved material: Microscopic inspection of the WAF showed no micro-dispersions or undissolved test material to be present.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Green algae
- Strain: CCAP 276/20
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa (CCAP), Dunstaffnage Marine Laboratory, Oban, Argyll, Scotland


ACCLIMATION
- Acclimation period: Not reported
- Culturing media and conditions: Cultures were maintained in the laboratory by periodic replenishment of culture media at a temperature of 20 +/- 1 °C under constant illumination (intensity of approximately 7000 lux) and constant aeration.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

Not reported

Hardness:

Not reported.

Test temperature:

24 +/- 1 °C

pH:

7.2 - 7.6

Dissolved oxygen:

Not reported.

Salinity:

Not relevant.

Nominal and measured concentrations:

100 mg/L (nominal)

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL glass conical flask containing 100 mL of test preparation, plugged with polyurethane foam bungs and incubated in an INFORS Multitron(R) Version 2 Incubator and constantly shaken at approximately 150 rpm
- Renewal rate of test solution: Test material was not renewed
- Initial cells density: 100000 cells/mL
- Control end cells density: 383000 cells/mL
- No. of vessels per concentration (replicates): Six (Two in the range-finding study)
- No. of vessels per control (replicates): Three (Two in the range-finding study)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reverse osmosis deionised water
- Total organic carbon: Not reported

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: Culture media was adjusted to pH 7.5 +/- 0.1 with 0.1N NaOH or HCl.
- Photoperiod: Continuous illumination
- Light intensity: Approximately 7000 lux

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: Coulter(R) Multisizer Particle Counter

TEST CONCENTRATIONS
- Test concentrations: 0 and 100 mg/L nominal WAF loading rate

RANGE FINDING STUDY
- Test concentrations: 0, 10 and 100 mg/L nominal WAF loading rate
- Results used to determine the conditions for the definitive study: No effects on growth were noted at 10 and 100 mg/L nominal WAF loading rates

Reference substance (positive control):

no

Remarks:

Not mandatory under the applied guideline.

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: WAF loading rate

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: WAF loading rate

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

other: WAF loading rate

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control: yes
- Observation of abnormalities: No abnormalities were detected in any of the control or test cultures at 72 hours
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: At the start and end of the mixing period and after the 1 hour settlement period, the 100 mg/L WAF loading rate was observed to have formed a clear, colourless media column with test material floating at the media surface. Microscopic examination of the WAF showed there to be no globules/micro-dispersions of the test material. At the start of the test, all control and test cultures were observed to be clear, colourless solutions. After the 72-hour test period, all control and test cultures were observed to be green dispersions.

Results with reference substance (positive control):

Not applicable.

Reported statistics and error estimates:

Statistical analysis of the growth rate data was conducted using a Student's t-test incorporating Bartlett's test for homogeneity of variance. There were no statistical differences (P >= 0.05) between the control and 100 mg/L WAF loading rate. Therefore, the NOEL was 100 mg/L nominal WAF loading rate.

Table 2: Effects of sulphonic acids, petroleum, calcium salts on growth of Scenedesmus subspicatus

Nominal loading rate (mg/L)	Replicate	pH		Cell density (cells/mL)				AUC at 72 hours	% Inhibition	Growth rate (0-72 hours)	% Inhibition
		0 hours	72 hours	0 hours	24 hours	48 hours	72 hours
Control	1	7.2	7.6	9.46 x10^3	5.76 x10^4	1.77 x10^5	3.76 x10^5	9.48 x10^6	-	0.051	-
	2	7.2	7.6	9.89 x10^3	5.76 x10^4	1.90 x10^5	3.92 x10^5
	3	7.2	7.6	9.49 x10^3	5.31 x10^4	1.47 x10^5	3.82 x10^5
	Mean	-	-	9.61 x10^3	5.61 x10^4	1.71 x10^5	3.83 x10^5
100	1	7.2	7.6	9.97 x10^3	5.96 x10^4	2.28 x10^5	4.14 x10^5	1.19 x10^7	[26]	0.052	[2]
	2	7.2	7.6	1.02 x10^4	6.62 x10^4	2.30 x10^5	3.97 x10^5
	3	7.2	7.6	9.80 x10^3	7.06 x10^4	2.22 x10^5	4.95 x10^5
	4	7.2	7.6	1.10 x10^4	7.55 x10^4	2.31 x10^5	4.77 x10^5
	5	7.2	7.6	1.03 x10^4	7.95 x10^4	2.33 x10^5	4.43 x10^5
	6	7.2	7.6	9.78 x10^3	7.29 x10^4	2.33 x10^5	4.38 x10^5
	Mean	-	-	1.02 x10^4	7.07 x10^4	2.29 x10^5	4.44 x10^5

AUC: Area under the curve

[Increase in growth compared to the control]

Comparison of areas under the growth curves

The area under the curve is taken to be an index of growth and was calculated using the following formula:

A=N₁- N₀x t₁+N₁+ N₂– 2N₀x (t₂- t₁) +N_n-1 + Nn – 2N₀x (t_n– t_n-1)

         2                       2                                           2

where

A = area

N₀= cell concentration at the start of the test

N₁= cell concentration at t1

N_n= cell concentration at tn

t₁= time of first measurement (hours from start)

t_n= time at nth measurement (hours from start)

Percentage inhibition of growth at each loading rate (IA) was calculated by comparing the area under the test curve (At) with that under the control curve (Ac) using the following equation:

IA =A_c- A_tx 100

         A_c

Comparison of growth rates

The average maximum growth rate (µ) for each culture was calculated from the straight section of the growth curve using the following equation:

µ =lnN_n- lnN₁

         t_n- t₁

where

µ = average maximum growth rate

N₀= cell concentration at the start of test

N_n= cell concentration at tn

t₀= time at first measurement (0 hours)

t_n= time of nth measurement (hours from the start)

Validity criteria fulfilled:

yes

Conclusions:

The study report is a valid guideline study with GLP compliance. The calcium sulfonate substance (CAS 61789-86-4) was not toxic to Selenastrum capricornutum at a loading rate up to 1000 mg/L nominal WAF, therefore the ErL50(96h) as well as the EbL50(96h) are considered to be > 100 mg/L.

Executive summary:

In a 72 hour toxicity study, the cultures of the green algae (Scenedesmus subspicatus CCAP 276/20) were exposed to sulphonic acids, petroleum, calcium salts (CAS 61789 -86 -4) at nominal concentrations of 0 and 100 mg/L nominal WAF loading rate under static conditions in accordance with the guideline OECD Guideline 201 (Alga, Growth Inhibition Test). The EL50 based biomass was >100 mg/L and the EL50 based on growth rate was >100 mg/L. There were no compound related phytotoxic effects.

 

This toxicity study is classified as acceptable and satisfies the guideline requirements for an algal growth inhibition study.

 

Results Synopsis

 

Test Organism: Scenedesmus subspicatus CCAP 276/20

Test Type: Static

72 hr EC50 (biomass): >100 mg/L WAF loading rate

72 hr EC50 (growth rate): >100 mg/L WAF loading rate

Description of key information

RA_CAS115733-09-0_EPA OTS 797.1050_Selenastrum capricornutum: NOEL: 1000 mg/L WAF, ErL50(72h) > 1000 mg/L. 
RA_CAS61789-86-4_OECD201_Scenedesmus subspicatus: NOEL: 100 mg/L WAF, ErL50(72h) > 100 mg/L. 

Key value for chemical safety assessment

EC50 for freshwater algae:

1 000 mg/L

EC10 or NOEC for freshwater algae:

1 000 mg/L

Additional information

General considerations

For the substance itself, no experimental data are available. The following read-across substances were used to fulfil the endpoint of toxicity to aquatic algae and cyanobacteria: Calcium sulfonate read-across substance CAS 115733 -90 -0 and Calcium sulfonate read-across substance CAS 61789 -86 -4. Since these substances belong to the same chemical group, aryl-alkyl sulfonates, the same behaviour and toxicity potential can be expected. Calcium sulfonate read-across substances (CAS 115733-09-0 and CAS 61789-86-4) were used to fulfil the requirements for the endpoint of toxicity to algae and cyanobacteria. Investigations concerning toxicity in marine algae were not performed. For the detailed procedure of the read-across principle and justifications, please refer to the separate Read-Across Statement attached to this Chemical Safety Report (Chemservice S.A., 2018).

Experimental results -Selenastrum capricornutum

In a key study the calcium sulfonate read-across substance (CAS 115733-09-0) was tested at nominal concentrations of 100, 300 and 1000 mg/L as WAF loading rates under static conditions (Ward, 1994). The study was performed according to US EPA Guideline OTS 797.1050. The algal population grew well during the test, since an average of 1,711,000 cells/mL was recorded in the control group; resulting in a doubling time of 13 h. The test vessels containing 1000 mg/L WAF were slightly cloudy at the start of the test and after 24 h exposure time. After 72 hours, biomass measurements were 80 %, 62 % and 70 % of the control at 100, 300 and 1000 mg/L WAF, respectively. At 96-hours, biomass measurements were 70 %, 66 % and 88 % of the control at 100, 300 and 1000 mg/L WAF, respectively. Based on these findings, the ErL50 (72h), ErL50(96h), EbL50(72h) as well as the EbL50(96h) value are considered to be > 1000 mg/L. The corresponding No Observed Effect Level (NOEL) amounts to 1000 mg/L WAF for each sampling period.

Experimental results -Scenedesmus subspicatus

In a 72 hour key study, the cultures of the green algae (Scenedesmus subspicatus) were exposed to sulphonic acids, petroleum, calcium salts (CAS 61789 -86 -4) at nominal concentrations of 0 and 100 mg/L nominal WAF loading rate under static conditions (limit test). The test was performed by Mead (2005) in accordance with the OECD Guideline 201 (Alga, Growth Inhibition Test). The EL50 based biomass was > 100 mg/L and the EL50 based on growth rate was > 100 mg/L. There were no compound related phytotoxic effects.