Reaction mass of m-terphenyl and o-terphenyl

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:

The experiment was performed in following time period: 2017-09-24 to 2017-12-23.

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:

Version of 2006

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

Version of 2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

The department of health of the government of the United Kingdom (2017-11-28)

Analytical monitoring:

yes

Remarks:

The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0, 24, 48 and 72 hours.

Details on sampling:

- Concentrations: Based on the results of the range-finding test the following test concentrations were assigned to the definitive test: 10, 18, 32, 56 and 100% v/v saturated solution. The geometric mean measured test concentrations were determined to be 0.0014, 0.0020, 0.0032, 0.015 and 0.027 mg/L.
- Sampling method: Samples were taken at 21, 50 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 10E3 cells/mL) was taken as the starting cell density.
- Sample storage conditions before analysis: Not relevant.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances) : Not relevant for the performed study.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata.
- Strain: CCAP 278/4.
- Source (laboratory, culture collection): 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, UK.
- Age of inoculum (at test initiation): Not reported.
- Method of cultivation: Master cultures were maintained in the laboratory by the periodic replenishment of culture medium. The master cultures were kept under constant agitation by orbital shaker (100 to 150 rpm) and constant illumination at 24 ±1 °C.

ACCLIMATION
- Acclimation period: Not reported.
- Culturing media and conditions (same as test or not): 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 10E3 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100 to 150 rpm) and constant illumination at 24 ±1°C until the algal cell density was approximately 10E4 to 10E5 cells/mL.
- Any deformed or abnormal cells observed: No such cells observed.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Remarks on exposure duration:

Samples were taken at 21, 50 and 72 hours to determine cell density.

Post exposure observation period:

No additional post exposure period implemented in test design.

Hardness:

Information not available.

Test temperature:

Incubation at at 24 ±1°C under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 to 730 nm). The temperature within the incubator was recorded daily.

pH:

pH at test begin (0 hours): 9.0 (control vessels), 8.2 (test concentration 0.0014 mg/L), 8.5 (test concentration 0.0020 mg/L), 7.8 (test concentration 0.0032 mg/L), 7.7 (test concentration 0.015 mg/L), 7.6 (test concentration 0.027 mg/L).
pH at test termination (after 72 hours): 7.1 (control vessels), 8.1 (test concentration 0.0014 mg/L), 8.3 (test concentration 0.0020 mg/L), 8.5 (test concentration 0.0032 mg/L), 8.7 (test concentration 0.015 mg/L), 8.6 (test concentration 0.027 mg/L).

Dissolved oxygen:

Information not available.

Salinity:

Not relevant for freshwater study.

Conductivity:

Information not available.

Nominal and measured concentrations:

The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0, 24, 48 and 72 hours. 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. Four additional samples of each test concentration were incubated alongside the test to provide samples for analysis at 24 and 48 hours. Duplicate samples were taken at 0, 24, 48 and 72 hours and stored frozen for further analysis if necessary. Analysis of the test preparations at 0 hours showed measured test concentrations to range from 0.0020 to 0.038 mg/L. A decline in measured test concentrations was observed after each 24-Hour period in the range of 0.0020 to 0.036 mg/L at 24 hours, 0.0017 to 0.033 mg/L at 48 hours and less than the limit of quantification (LOQ), determined to be 0.00033 mg/L, to 0.0028 mg/L at 72 hours. Given this decline in measured test concentrations, it was considered appropriate to calculate the results based on the geometric mean measured test concentration only in order to give a “worst case” analysis of the data. The geometric mean measured test concentrations were determined to be 0.0014, 0.0020, 0.0032, 0.015 and 0.027 mg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL glass conical flasks were used.
- Type (delete if not applicable): Closed. Flasks were plugged with polyurethane foam bungs to reduce evaporation.
- Material, size, headspace, fill volume: glass, 250 mL volume capacity, filled with 100 mL test solution.
- Aeration: No.
- Type of flow-through (e.g. peristaltic or proportional diluter): Test was performed under static conditions.
- Renewal rate of test solution (frequency/flow rate): Not applicable.
- Initial cells density: Pre-culture conditions gave an algal suspension in log phase growth characterized by a cell density of 2.63 x 10E5 cells per mL. Inoculation of 2 liters of test medium with 38.0 mL of this algal suspension gave an initial nominal cell density of 5.00 x 10E3 cells per mL and had no significant dilution effect on the final test concentration.
- Control end cells density: In control vessels cell density was in the range of 1.38E6 to 1.60E6 (n=6) at test termination.
- No. of organisms per vessel: Not vapplicable to algae study.
- No. of vessels per concentration (replicates): Three flasks each containing 100 mL were used for each treatment group.
- No. of vessels per control (replicates): Six flasks each containing 100 mL of solution were used for the control group
- No. of vessels per vehicle control (replicates): No vehicle used.

GROWTH MEDIUM
- Standard medium used: Yes. The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture.
- Detailed composition if non-standard medium was used: NaNO3: 25.5 mg/L, MgCl2 x 6H2O: 12.16 mg/L, CaCl2 x 2H2O: 4.41 mg/L, MgSO4 x 7H2O: 14.6 mg/L,
K2HPO4: 1.044 mg/L, NaHCO3: 15.0 mg/L, H3BO3: 0.186 mg/L, MnCl2 x 4H2O: 0.415 mg/L, ZnCl2: 0.00327 mg/L, FeCl3.6H2O: 0.160 mg/L, CoCl2 x 6H2O: 0.00143 mg/L,
Na2MoO4 x 2H2O: 0.00726 mg/L, CuCl2 x 2 H2O: 0.000012 mg/L, Na2EDTA x 2H2O: 0.30 mg/L.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Information not available.
- Total organic carbon: Information not available.
- Particulate matter: Information not available.
- Metals: Information not available.
- Pesticides: Information not available.
- Chlorine: Information not available.
- Alkalinity: Information not available.
- Ca/Mg ratio: Information not available.
- Conductivity: Information not available.
- Culture medium different from test medium: No, same culture medium was used.
- Intervals of water quality measurement: The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0, 24, 48 and 72 hours. The temperature within the incubator was recorded daily. The pH of the control and each test preparation was determined at initiation of the test and after 72 hours exposure. The appearance of the test media was recorded daily.

OTHER TEST CONDITIONS
- Sterile test conditions: No
- Adjustment of pH: pH was not adjusted.
- Photoperiod: Continuous illumination during incubation period.
- Light intensity and quality: Light intensity approximately 7000 lux, provided by warm white lighting (380 to 730 nm)
- Salinity (for marine algae): Not relevant for freshwater study.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Samples were taken at 21, 50 and 72 hours and the cell densities determined. To determine the potential effect of the test item on the appearance of algal cells, a sample was removed from each test and control culture (replicates pooled) at the end of the test. The shape and size of the algal cells was inspected microscopically and any abnormalities recorded.
- Determination of cell concentrations: Samples were taken at 21, 50 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 10E3 cells/mL) was taken as the starting cell density.
- Chlorophyll measurement: Not performed.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: No specific spacing factor applied.
- Justification for using less concentrations than requested by guideline: Not relevant.
- Range finding study: The test concentrations to be used in the definitive test were determined by a preliminary range-finding test. After 72 hours the cell density of each flask was 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. Only concentrations within the range to be used for the definitive test were analyzed.
- Test concentrations: The range-finding test was conducted by exposing Pseudokirchneriella subcapitata cells to a series of nominal test concentrations of 1.0, 10 and 100% v/v saturated solution for a period of 72 hours. Prior to use the test item was heated to 60 ºC in order to aid handling.
- Results used to determine the conditions for the definitive study: Yes, based on the results of the range-finding test the following test concentrations were assigned to the definitive test: 10, 18, 32, 56 and 100% v/v saturated solution.

Reference substance (positive control):

yes

Remarks:

A positive control (Envigo study number RD71YQ) used potassium dichromate as the reference item. The positive control was conducted between 06 November 2017 and 09 November 2017.

Duration:

72 h

Dose descriptor:

EC50

Remarks:

Response variable: Yield

Effect conc.:

0.027 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Key result

Duration:

72 h

Dose descriptor:

EC50

Remarks:

Response variable: Growth rate

Effect conc.:

> 0.027 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other:

Remarks:

It was not possible to calculate ErC20 or ErC50 values as at the maximum attainable test concentration of 0.027 mg/L, no more than 18% inhibition of growth rate occurred.

Duration:

72 h

Dose descriptor:

NOEC

Remarks:

Response variable: Growth rate

Effect conc.:

0.015 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Remarks:

Response variable: Growth rate

Effect conc.:

0.027 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Remarks:

Response variable: Yield

Effect conc.:

0.015 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

LOEC

Remarks:

Response variable: Yield

Effect conc.:

27 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

EC10

Remarks:

Response variable: Growth rate

Effect conc.:

0.026 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC20

Remarks:

Response variable: Growth rate

Effect conc.:

> 0.027 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: It was not possible to calculate ErC20 or ErC50 values as at the maximum attainable test concentration of 0.027 mg/L, no more than 18% inhibition of growth rate occurred.

Duration:

72 h

Dose descriptor:

EC10

Remarks:

Response variable: Yield

Effect conc.:

0.025 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

EC20

Remarks:

Response variable: Yield

Effect conc.:

0.026 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Details on results:

- Exponential growth in the control (for algal test): Yes
- Observation of abnormalities (for algal test): All test and control cultures were inspected microscopically at 72 hours. There were no abnormalities detected in any of the control or test cultures.
- Unusual cell shape: No.
- Colour differences: At the start of the test all control and test cultures were observed to be clear colorless solutions. After the 72-Hour test period all control, 0.0014, 0.0020, 0.0032 and 0.015 mg/L test cultures were observed to be green dispersions whilst the 0.027 mg/L test cultures were observed to be slightly pale green dispersions.
- Flocculation: No.
- Adherence to test vessels: No.
- Aggregation of algal cells: No.
- Other: The pH value of the control cultures was observed to decrease from pH 9.0 at 0 hours to pH 7.1 at 72 hours. The high 0-Hour measured value was considered to be due to failure to allow the pH probe to equilibrate following the pH 10 confirmatory check. Given this, the pH deviation between 0 and 72 hours was greater than 1.5 units. This was considered to have had no adverse effect on the outcome of the test as all other validity criteria were met.
- Any stimulation of growth found in any treatment: No stimulation observed.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No.
- Effect concentrations exceeding solubility of substance in test medium: Not relevant.

Results with reference substance (positive control):

- Results with reference substance valid? Yes. A positive control (Envigo study number RD71YQ) 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.
- EC50: ErC50 (0 to 72 hour) : 1.6 mg/L; 95% confidence limits 1.4 to 1.8 mg/L. EyC50 (0 to 72 hour) : 0.77 mg/L; 95% confidence limits 0.68 to 0.87 mg/L.
The results from the positive control with potassium dichromate were within the normal ranges for this reference item.
- Other: No Observed Effect Concentration based on growth rate: 0.25 mg/L. No Observed Effect Concentration based on yield: 0.25 mg/L
Lowest Observed Effect Concentration based on growth rate: 0.50 mg/L. Lowest Observed Effect Concentration based on yield: 0.50 mg/L

Reported statistics and error estimates:

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: A Multiple Comparison Procedure for Comparing Several Treatments With a Control. J Am Stat Assoc 50, 1096-1121.). All statistical analyses were performed using the SAS computer software package (SAS, 1999 - 2001). There were no statistically significant decreases in growth rate between the control, 0.0014, 0.0020, 0.0032 and 0.015 mg/L test concentrations (P≥0.05), however the 0.027 mg/L test concentration was significantly different (P<0.05) and, therefore the NOEC based on growth rate was 0.015 mg/L. Correspondingly the Lowest Observed Effect Concentration (LOEC) based on growth rate was 0.027 mg/L.

Table 2: Results of the 72h aquatic toxicity study with algae (Pseudokirchneriella subcapitata):

Response variable	EC10 (mg/L)	EC20 (mg/L)	EC50 (mg/L)	NOEC (mg/L)	LOEC (mg/L)
Growth rate	0.026	>0.027*	>0.027*	0.015	0.027
Yield	0.025	0.026	0.027	0.015	0.027

*: It was not possible to calculate ErC20 or ErC50 values as at the maximum attainable test concentration of 0.027 mg/L, no more than 18% inhibition of growth rate occurred.

Validity criteria fulfilled:

yes

Conclusions:

A study according to OECD Guideline 201 was performed to determine the aquatic toxicity of the test compound to green alga Pseudokirchneriella subcapitata.
Results for growth rate (72 h): EC50: >0.027 mg/L*, EC20>0.027 mg/L*, EC10: 0.026 mg/L, LOEC: 0.015 mg/L, NOEC: 0.027 mg/L.
*:It was not possible to calculate ErC20 or ErC50 values as at the maximum attainable test concentration of 0.027 mg/L, no more than 18% inhibition of growth rate occurred.
Results for yield (72 h): EC50: 0.027 mg/L, EC20>0.026 mg/L, EC10: 0.025 mg/L, LOEC: 0.015 mg/L, NOEC: 0.027 mg/L.

Executive summary:

A study was performed to assess the effect of the test item on the growth of the green alga Pseudokirchneriella subcapitata. The method followed that described in the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of Commission Regulation (EC) No 761/2009. The study was performed according to GLP.

Preliminary solubility work conducted indicated that it was not possible to obtain a testable solution of the test item using traditional methods of preparation e.g. ultra-sonication and high shear mixing. A preliminary media preparation trial indicated that a dissolved test item concentration of approximately 0.062 mg/L was obtained from a saturated solution method of preparation indicating this to be the limit of water solubility of this item under test conditions.

Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to solutions of the test item at nominal concentrations of 10, 18, 32, 56 and 100% v/v saturated solution (3 replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ±1°C. The test item solutions were prepared by stirring an excess (50 mg/L) of test item in culture medium using a propeller stirrer at approximately 1500 rpm for 48 hours. After the stirring period any undissolved test item was removed by filtration (0.2μm Sartorius Sartopore filter, first approximate 2 liters discarded in order to pre-condition the filter) to produce a 100% v/v saturated solution of the test item. This saturated solution was then further diluted as necessary, to provide the remaining test groups. Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter® Multisizer Particle Counter. The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0, 24, 48 and 72 hours

Results: Analysis of the test preparations at 0 hours showed measured test concentrations to range from 0.0020 to 0.038 mg/L. A decline in measured test concentrations was observed after each 24-Hour period in the range of 0.0020 to 0.036 mg/L at 24 hours, 0.0017 to 0.033 mg/L at 48 hours and less than the limit of quantification (LOQ), determined to be 0.00033 mg/L, to 0.0028 mg/L at 72 hours. Given this decline in measured test concentrations it was considered appropriate to calculate the results based on the geometric mean measured test concentration only in order to give a “worst case” analysis of the data. The geometric mean measured test concentrations were determined to be 0.0014, 0.0020, 0.0032, 0.015 and 0.027 mg/L.

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

Results of the 72h aquatic toxicity study with algae (Pseudokirchneriella subcapitata):

Response variable	EC10 (mg/L)	EC20 (mg/L)	EC50 (mg/L)	NOEC (mg/L)	LOEC (mg/L)
Growth rate	0.026	>0.027*	>0.027*	0.015	0.027
Yield	0.025	0.026	0.027	0.015	0.027

*:It was not possible to calculate ErC20 or ErC50 values as at the maximum attainable test concentration of 0.027 mg/L, no more than 18% inhibition of growth rate occurred.

Description of key information

OECD 201, GLP, P. subcapitata, static, EC50 (72 h) > 0.027 mg/L

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

0.026 mg/L

Additional information

A study was performed to assess the effect of the test item on the growth of the green alga Pseudokirchneriella subcapitata. The method followed that described in the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of Commission Regulation (EC) No 761/2009.

Preliminary solubility work conducted indicated that it was not possible to obtain a testable solution of the test item using traditional methods of preparation e.g. ultra-sonication and high shear mixing. A preliminary media preparation trial indicated that a dissolved test item concentration of approximately 0.062 mg/L was obtained from a saturated solution method of preparation indicating this to be the limit of water solubility of this item under test conditions.

Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to solutions of the test item at nominal concentrations of 10, 18, 32, 56 and 100% v/v saturated solution (3 replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1°C. The test item solutions were prepared by stirring an excess (50 mg/L) of test item in culture medium using a propeller stirrer at approximately 1500 rpm for 48 hours. After the stirring period any undissolved test item was removed by filtration (0.2μm Sartorius Sartopore filter, first approximate 2 liters discarded in order to pre-condition the filter) to produce a 100% v/v saturated solution of the test item. This saturated solution was then further diluted as necessary, to provide the remaining test groups. Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter® Multisizer Particle Counter. The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0, 24, 48 and 72 hours.

Results: Analysis of the test preparations at 0 hours showed measured test concentrations to range from 0.0020 to 0.038 mg/L. A decline in measured test concentrations was observed after each 24-Hour period in the range of 0.0020 to 0.036 mg/L at 24 hours, 0.0017 to 0.033 mg/L at 48 hours and less than the limit of quantification (LOQ), determined to be 0.00033 mg/L, to 0.0028 mg/L at 72 hours. Given this decline in measured test concentrations it was considered appropriate to calculate the results based on the geometric mean measured test concentration only in order to give a “worst case” analysis of the data. The geometric mean measured test concentrations were determined to be 0.0014, 0.0020, 0.0032, 0.015 and 0.027 mg/L.

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

Results of the 72h aquatic toxicity study with algae (Pseudokirchneriella subcapitata):

Response variable	EC10 (mg/L)	EC20 (mg/L)	EC50 (mg/L)	NOEC (mg/L)	LOEC (mg/L)
Growth rate	0.026	>0.027*	>0.027*	0.015	0.027
Yield	0.025	0.026	0.027	0.015	0.027

*:It was not possible to calculate ErC20 or ErC50 values as at the maximum attainable test concentration of 0.027 mg/L, no more than 18% inhibition of growth rate occurred.