Sulfonic acids, shale-oil, sodium salts

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:

initiated: 2016-08-22, experimental: 2016-12-06 to 2017-02-23

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

recommended study/method

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2006

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

MHRA, date of issue: 28/10/2016

Specific details on test material used for the study:

technical grade: aqueous solution (31% dry matter in water)

Analytical monitoring:

yes

Details on sampling:

METHOD: Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter® Multisizer Particle Counter.

RANGE-FINDING TEST: At the start of the range-finding test a sample of each test and control culture was removed and the cell density determined using a Coulter® Multisizer Particle Counter. A sample of each test concentration was taken for chemical analysis at 0 and 72 hours in order to determine the stability of the test item under test conditions. All samples were stored frozen prior to analysis.

TEST ORGANISM OBSERVATION: Samples were taken at 24, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter.
VERIFICATION OF TEST CONCENTRATIONS: Samples were taken from the control and each test group from the bulk test preparation at 0 hours and from the pooled replicates at 72 hours for quantitative analysis. All samples were stored frozen prior to analysis. Duplicate samples were taken at 0 and 72 hours and stored frozen for further analysis if necessary.

Vehicle:

no

Details on test solutions:

The test item contains 31% active ingredient. The active ingredient concentration in the test samples was determined by high performance liquid chromatography with tandem quadrapole mass spectrometery (HPLC-MS/MS) using an external standard. The active ingredient gave a chromatographic profile consisting of several peaks.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM:
-Common name: green algae
- Strain: Pseudokirchneriella subcapitata CCAP 278/4
- Source: Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland

OBSERVATION:
Samples were taken at 24, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter. Three determinations were made for each sample. The nominally inoculated cell concentration (5.00 x 103 cells/mL) was taken as the starting cell density.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Remarks on exposure duration:

The flasks were plugged with polyurethane foam bungs and incubated at 24 ± 1 °C under continuous illumination (intensity approx. 7000 lux) provided by warm white lighting (380 – 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.

Hardness:

NDA

Test temperature:

24 ± 1 °C

pH:

7.9 (0 h) - 8.5 (72 h)

Dissolved oxygen:

NDA

Salinity:

NDA

Nominal and measured concentrations:

RANGE-FINDING TEST: 0.10, 1.0, 10, 100 mg ai/L
DEFINITIVE TEST: 1.0, 3.2, 10, 32, 100 mg ai/L

Details on test conditions:

TEST ITEM:
Tiroler Steinöl sulfoniert D90 (dark brown liquid). A Certificate of Analysis was supplied by the Sponsor.

TEST SYSTEM:
The test was carried out using Pseudokirchneriella subcapitata strain CCAP 278/4. Liquid cultures of Pseudokirchneriella subcapitata were obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland. Master cultures were maintained in the laboratory by the periodic replenishment of culture medium (Section 3.3). The master cultures were maintained in the laboratory under constant aeration and illumination at 21 ± 2 °C.
Prior to the start of the test sufficient master culture was added to approximately 100 mL volumes of culture media contained in conical flasks to give an initial cell density of approximately 103 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100 – 150 rpm) and constant illumination at 24 ± 1 °C until the algal cell density was approximately 104 – 105 cells/mL.
A positive control (Envigo Study Number FP48BQ) used potassium dichromate as the reference item. Details of the positive control are given in Annex 2. The positive control was conducted between 28 November 2016 and 01 December 2016.

CULTUR MEDIUM:
The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture.
Macro-nutritiens:
NaNO3 25.5 mg/L
NaHCO3 15.0 mg/L
MgSO4.7H2O 14.6 mg/L
MgCl2.6H2O 12.16 mg/L
CaCl2.2H2O 4.41 mg/L
K2HPO4 1.044 mg/L
Trace elements:
MnCl2.4H2O 0.415 mg/L
Na2EDTA.2H2O 0.30 mg/L
H3BO3 0.186 mg/L
FeCl3.6H2O 0.160 mg/L
Na2MoO4.2H2O 0.00726 mg/L
ZnCl2 0.00327 mg/L
CoCl2.6H2O 0.00143 mg/L
CuCl2.2H2O 0.000012 mg/L

The culture medium was prepared using reverse osmosis purified deionized water* and the pH adjusted to 7.5 with 0.1N NaOH or HCl.

EXPOSURE CONDITIONS:
As in the range-finding test 250 mL glass conical flasks were used. Six flasks each containing 100 mL of test preparation were used for the control and three flasks each containing 100 mL were used for each treatment group. The control group was maintained under identical conditions but not exposed to the test item.
Pre-culture conditions gave an algal suspension in log phase growth characterized by a cell density of 5.15 x 105 cells per mL. Inoculation of 450 mL of test medium with 4.4 mL of this algal suspension gave an initial nominal cell density of 5.00 x 103 cells per mL and had no significant dilution effect on the final test concentration. The flasks were plugged with polyurethane foam bungs and incubated (INFORS Multitron Version 2 incubator) at 24 ± 1 °C under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.

WATER QUALITY CRITERIA:
The pH of the control and each test preparation was determined at initiation of the test and after 72 hours exposure. The pH was measured using a Hach HQ30d Flexi handheld meter. The temperature within the incubator was recorded daily. The appearance of the test media was recorded daily.

VERIFICATION OF TEST CONCENTRATIONS:
Samples were taken from the control and each test group from the bulk test preparation at 0 hours and from the pooled replicates at 72 hours for quantitative analysis. All samples were stored frozen prior to analysis. Duplicate samples were taken at 0 and 72 hours and stored frozen for further analysis if necessary.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 72 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

44 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

24 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

24 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

72 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

72 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

RANGE-FINDING TEST:
The cell densities and percentage inhibition of growth values from the exposure of Pseudokirchneriella subcapitata to the test item during the range-finding test are:
- Cell densities (cells per ml), 0 h: 3.79E+3 to 4.83E+3
- Cell densities (cells per ml), 72 h: 1.13E+5 to 5.93E+5
- Inhibition values (%, growth rate): 0 - 33
- Inhibition values (%, yield): 2 - 78

The results showed no effect on growth at the test concentrations of 0.10 and 1.0 mg ai/L. However, growth was observed to be reduced at 10 and 100 mg ai/L.
Based on this information test concentrations of 1.0, 3.2, 10, 32 and 100 mg ai/L were selected for the definitive test. Chemical analysis of the test preparations at 0 and 72 hours (see Annex 4) showed near nominal concentrations were obtained.

DEFINITIVE TEST:
Verification of Test Concentrations - Analysis of the test preparations at 0 hours (see Annex 4) showed measured test concentrations to range from 80% to 92% of nominal. A decline in measured concentrations was observed at 72 hours in the range of 63% to 74% of nominal. Current regulatory advice is that in cases where a decline in measured concentrations is observed, geometric mean measured concentrations should be used for calculating EC50 values. It was therefore considered justifiable to base the results on the geometric mean measured test concentrations in order to give a “worst case” analysis of the data.

Growth Data - Cell density values determined at each sampling time and pH values at 0 and 72 hours are given in Table 2. Daily specific growth rates for the control cultures are given in Table 3. Growth rate and yield values for the control and test cultures after 72 hours and percentage inhibition values are:
- Inhibition values, 0 - 72 h (%, growth rate): 0 - 24
- Inhibition values, 0 - 72 h (%, yield): 1 - 65

Accordingly the following results were determined from the data based on the geometric mean measured test concentrations:
Inhibition of growth rate
ErC10 (0 - 72 h) : 38 mg ai/L
ErC20 (0 - 72 h) : 67 mg ai/L
ErC50 (0 - 72 h) : >72 mg ai/L
where ErCx is the test concentration that reduced growth rate by x%.

It was not possible to calculate an ErC50 value as no concentration tested resulted in greater than 50% inhibition of growth rate. Statistical analysis of the growth rate data was carried out for the control and all test concentrations using one way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett, 1955). There were no statistically significant differences (P≥0.05), between the control, 0.79, 2.4, 8.0 and 24 mg ai/L test concentrations however the 72 mg ai/L test concentration was significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on growth rate was 24 mg ai/L. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on growth rate was 72 mg ai/L.

Inhibition of Yield
EyC10 (0 - 72 h) : 15 mg ai/L
EyC20 (0 - 72 h) : 20 mg ai/L
EyC50 (0 - 72 h) : 44 mg ai/L
where EyCx is the test concentration that reduced yield by x%.

Statistical analysis of the yield data was carried out as in Section 6.2.3. There were no statistically significant differences (P≥0.05), between the control, 0.79, 2.4, 8.0 and 24 mg ai/L test concentrations however the 72 mg ai/L test concentration was significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on yield was 24 mg ai/L. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on yield was 72 mg ai/L.

From the data given, it is clear that the growth rate (r) and yield (y) of Pseudokirchneriella subcapitata (CCAP 278/4) were affected by the presence of the test item over the 72-Hour exposure period.

Results with reference substance (positive control):

POSITIVE CONTROL:
A positive control (Envigo Study Number FP48BQ) used potassium dichromate as the reference item at concentrations of 0.25, 0.50, 1.0, 2.0 and 4.0 mg/L. Exposure conditions and data evaluation for the positive control were similar to those in the definitive test. Exposure of Pseudokirchneriella subcapitata (CCAP 278/4) to the reference item gave the following results:

ErC50 (0 – 72 h) : 1.4 mg/L; 95% confidence limits 1.2 – 1.5 mg/L
EyC50 (0 – 72 h) : 0.60 mg/L; 95% confidence limits 0.52 – 0.69 mg/L

No Observed Effect Concentration (NOEC) based on growth rate: 0.25 mg/L
No Observed Effect Concentration (NOEC) based on yield: 0.25 mg/L
Lowest Observed Effect Concentration (LOEC) based on growth rate: 0.50 mg/L
Lowest Observed Effect Concentration (LOEC) based on yield: 0.50 mg/L

The results from the positive control with potassium dichromate were within the normal ranges for this reference item.

Reported statistics and error estimates:

All statistical analyses were performed using the SAS computer software package (SAS, 1999 - 2001).

VALIDATION CRITERIA:
a) The following data show that the cell concentration of the control cultures increased by a factor of 122 after 72 hours. This increase was in line with the OECD Guideline that states the enhancement must be at least by a factor of 16 after 72 hours.
- Nominal cell density of control at 0 hours : 5.00 x 103 cells per mL
- Mean cell density of control at 72 hours : 6.11 x 105 cells per mL

b) The mean coefficient of variation for section by section specific growth rate for the control cultures was 14% and hence satisfied the validation criterion given in the OECD Guideline which states the mean must not exceed 35%.

c) The coefficient of variation for average specific growth rate for the control cultures over the test period (0 – 72 h) was 4% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.

Observations on Test Item Solubility

At the start of the test all control and 1.0 mg ai/L test cultures were observed to be clear colorless solutions. The 3.2, 10, 32 and 100 mg ai/L test cultures were observed to range from extremely pale brown solutions through to brown solutions, the intensity of the coloration increasing with increased test concentration. After the72-Hour test period replicate R3 of the control was observed to be a bright green dispersion, all other control replicates and the test replicates at 1.0, 3.2 and 10 mg ai/L were observed to be green dispersions. The 32 mg ai/L test cultures were observed to be green/yellow dispersions whilst the 100 mg ai/L test cultures were observed to be yellow dispersions.

Validity criteria fulfilled:

yes

Remarks:

criteria a), b) and c) fulfilled

Conclusions:

The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated over a 72-Hour period and based on the geometric mean measured test concentrations gave the following results:

Growth Rate:
- EC50 > 72 mg/L
- NOEC = 24 mg/L
- LOEC = 72 mg/L

Yield:
- EC50 = 44 mg ai/L
- NOEC = 24 mg ai/L
- LOEC = 72 mg ai/L

Executive summary:

A study was performed to assess the effect of sulfonic acids, shale oil, sodium salts on the growth of the green alga Pseudokirchneriella subcapitata. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" and performed according to GLP rules.

Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to an aqueous solution of the test item at nominal concentrations of 1.0, 3.2, 10, 32 and 100 mg active ingredient (ai)/L (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1 °C.

Analysis of the test preparations at 0 hours showed measured test concentrations to range from 80% to 92% of nominal. A decline in measured concentrations was observed at 72 hours in the range of 63% to 74% of nominal and hence it was considered appropriate to calculate the results based on the geometric mean measured test concentrations which were determined to be 0.79, 2.4, 8.0, 24 and 72 mg ai/L.

Exposure of Pseudokirchneriella subcapitata to the test item gave the following results based on the geometric mean measured test concentrations:

Growth Rate:

 - EC50  > 72 mg ai/L

 - NOEC = 24 mg ai/L

 - LOEC = 72 mg ai/L

Yield:

 - EC50 = 44 mg ai/L

 - NOEC = 24 mg ai/L

 - LOEC = 72 mg ai/L

Description of key information

The following data show that the cell concentration of the control cultures increased by a factor of 122 after 72 hours. This increase was in line with the OECD Guideline that states the enhancement must be at least by a factor of 16 after 72 hours.

- Nominal cell density of control at 0 hours : 5.00 x 103 cells per mL

- Mean cell density of control at 72 hours : 6.11 x 105 cells per mL

The mean coefficient of variation for section by section specific growth rate for the control cultures was 14% and hence satisfied the validation criterion given in the OECD Guideline which states the mean must not exceed 35%.

The coefficient of variation for average specific growth rate for the control cultures over the test period (0 – 72 h) was 4% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.

Key value for chemical safety assessment

EC50 for freshwater algae:

72 mg/L

EC10 or NOEC for freshwater algae:

24 mg/L

Additional information

Observations on Cultures

All test and control cultures were inspected microscopically at 72 hours. After 72 hours there were no abnormalities detected in the control or test cultures at 1.0, 3.2, 10 and 32 mg ai/L, however some mutated/misshapen cells were observed to be present in the test cultures at 100 mg ai/L.

Water Quality Criteria

The pH values of the control and each test preparation are given in Table 2. Temperature was maintained at 24 ± 1 ºC throughout the test.

The pH value of the control cultures (see Table 2) was observed to increase from pH 7.9 at 0 hours to pH 8.5 at 72 hours. The pH deviation in the control cultures was less than 1.5 pH units after 72 hours and therefore was within the limits given in the Test Guidelines.

Observations on Test Item Solubility

At the start of the test all control and 1.0 mg ai/L test cultures were observed to be clear colorless solutions. The 3.2, 10, 32 and 100 mg ai/L test cultures were observed to range from extremely pale brown solutions through to brown solutions, the intensity of the coloration increasing with increased test concentration. After the72-Hour test period replicate R3 of the control was observed to be a bright green dispersion, all other control replicates and the test replicates at 1.0, 3.2 and 10 mg ai/L were observed to be green dispersions. The 32 mg ai/L test cultures were observed to be green/yellow dispersions whilst the 100 mg ai/L test cultures were observed to be yellow dispersions.