Tar acids, xylenol fraction

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:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

according to guideline

Guideline:

other: DEV L9

Principles of method if other than guideline:

cell multiplication inhibition test

GLP compliance:

no

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

a stock solution was prepared; to achive the test concentrations a defined amount of the stock solution was dissolved

Test organisms (species):

Microcystis aeruginosa

Details on test organisms:

blue-green algae = cyanobacteria

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

8 d

Test temperature:

27 °C

pH:

initial pH 7.0

Duration:

8 d

Dose descriptor:

other: TGK

Effect conc.:

13 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: cell multiplication

In the cited literature, the experiments were conducted with the species Microcystis aeruginosa and Scenedesmus quadricauda. In the OECD/ICCA HPV programm it was stated that it is unclear whether the algae S. quadricauda are within the exponential growth throughout the whole exposure period of 8 days. Accordingly, the study was assessed to be not reliable (reliability 3). However, the bluegreen algae Microcystis aeruginosa have a lower rate of reproduction compared to green algae and are still in the exponential growth rate under the experimental conditions after 8 d.

Executive summary:

The toxicity of m-cresol to the blue-green algae Microcystis aeruginosa (Cyanaophyceae) was determined in a cell multiplication inhibition test according to the guideline DEV L9. The relevant 8d-NOEC (EC3) for growth rate is 13 mg/L (Bringmann 1975, Bringmann and Kuehn 1976).

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: DEV L9

Principles of method if other than guideline:

cell multiplication inhibition test

GLP compliance:

no

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

a stock solution was prepared; to achive the test concentrations a defined amount of the stock solution was dissolved

Test organisms (species):

Microcystis aeruginosa

Details on test organisms:

blue-green algae = cyanobacteria

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

8 d

Test temperature:

27 °C

pH:

initial pH 7.0

Duration:

8 d

Dose descriptor:

other: TGK

Effect conc.:

6.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: cell multiplication

In the cited literature, the experiments were conducted with the species Microcystis aeruginosa and Scenedesmus quadricauda. In the OECD/ICCA HPV programm it was stated that it is unclear whether the algae S. quadricauda are within the exponential growth throughout the whole exposure period of 8 days. Accordingly, the study was assessed to be not reliable (reliability 3). However, the bluegreen algae Microcystis aeruginosa have a lower rate of reproduction compared to green algae and are still in the exponential growth rate under the experimental conditions after 8 d.

Validity criteria fulfilled:

not specified

Executive summary:

The toxicity of o-cresol to the blue-green algae Microcystis aeruginosa (Cyanaophyceae) was determined in a cell multiplication inhibition test according to the guideline DEV L9. The relevant 8d-NOEC (EC3) for growth rate is 6.8 mg/L (Bringmann 1975, Bringmann and Kuehn 1976).

Reference 3

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

no guideline followed

Principles of method if other than guideline:

Two methods applied:
1) Algal cultures were grown in test tubes for a period of 72 hours. The toxicity was evaluated by measuring changes in chlorophyll content of the algal suspensions every 24 hours.
2) The Warburg manometric technique was used to measure the photosynthetic gas exchange that occurs when a carbonate-bicarbonate buffer is used as the suspending fluid.

GLP compliance:

no

Analytical monitoring:

no

Test organisms (species):

Chlorella pyrenoidosa

Details on test organisms:

An Emerson strain of bacteria-free, experimentally reproducible cultures of Chlorella pyrenoidosa was obtained from Dr. Jack E. Myers, Professor of Zoology and Botany and Director, Laboratory of Algal Physiology, The University of Texas. These algal cultures had a density of 1.0 gm/L dry weight, or equivalent to 3.8 cmm/mL packed
cell volume, and had a chlorophyll content of 38 mg/L.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

25 °C

pH:

7

Nominal and measured concentrations:

control, 50, 100, 250, 500, 750, and 1000 mg/L (nominal)

Details on test conditions:

TEST SYSTEM
- Test vessel: Aquarium constructed of lucite; 48 inches long, 12 inches wide, 6.75 inches deep. To hold the test tubes which were used as the actual test vessels, two masonite boards with matched and equally spaced holes were positioned near the top and the bottom of the aquarium. A test tube contained 15 mL of inorganic culture medium with a predetermined amount of the tested organic compound and 5 mL of algal source culture suspension making a total volume of 20 mL. Covered with caps
- Aeration: A stream of 5% carbon dioxide in air gas mixture was supplied to provide the inorganic carbon souree and also to keep the algal cells in suspension
- No. of vessels per concentration (replicates): three test tubes during the screening tests, five tubes during the final testso

GROWTH MEDIUM
- Knop's solution, including the Hutner-EDTA microelement system


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Tap water

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: yes, with KOH
- Photoperiod: continuous
- Light intensity and quality: four 200-watt fluorescent lamps with attached aluminum reflectors; intensity of 550 to 650 foot-candles
- Temperature: Tap water with a constant head was continuously circulated through the aquarium to maintain a relatively constant temperature.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: A Beckman Spectrophotometer, Model DB, was used for all chlorophyll analysis. This spectrophotometer is a double beam instrument
for making transmittance and absorbance measurements in the 205 to 770 µm wavelength range
- Chlorophyll measurement: Measuring changes in chlorophyll content of the algal suspensions every 24 hours.
- Other: The Warburg manometric technique was used to measure the photosynthetic gas exchange that occurs when a carbonate-bicarbonate buffer is used as the suspending fluid

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

50 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: chlorophyll reduction

Validity criteria fulfilled:

not applicable

Reference 4

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Qualifier:

according to guideline

Guideline:

other: DIN 38412 part 9 (draft standard)

Deviations:

yes

Remarks:

250 ml glass-stoppered bottles 48h

GLP compliance:

no

Analytical monitoring:

no

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

strain no. 8681 SAG

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test temperature:

24 ± 1 °C

Nominal and measured concentrations:

Test substance concentrations 0.8 - 100 mg/L, dilution series 1:2

Details on test conditions:

preliminary culture 10E5 cells/L
irradiance 17.0 W/m2

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

48 h

Dose descriptor:

EC10

Effect conc.:

2.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

7.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

48 h

Dose descriptor:

EC10

Effect conc.:

4.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

21 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Validity criteria fulfilled:

not specified

Executive summary:

The toxicity of p-cresol to the algae Desmodesmus subspicatus (former name Scenedesmus subspicatus) was determined according to DIN 38412 part 9 (draft standard for cell multiplication inhibition). Based on nominal concentrations a 48 h-EC50 of 21 mg/l and an EC10 of 4.6 mg/l (both related to growth rate) were determined.

Reference 5

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

no guideline followed

Principles of method if other than guideline:

Two methods applied:
1) Algal cultures were grown in test tubes for a period of 72 hours. The toxicity was evaluated by measuring changes in chlorophyll content of the algal suspensions every 24 hours.
2) The Warburg manometric technique was used to measure the photosynthetic gas exchange that occurs when a carbonate-bicarbonate buffer is used as the suspending fluid.

GLP compliance:

no

Analytical monitoring:

no

Test organisms (species):

Chlorella pyrenoidosa

Details on test organisms:

An Emerson strain of bacteria-free, experimentally reproducible cultures of Chlorella pyrenoidosa was obtained from Dr. Jack E. Myers, Professor of Zoology and Botany and Director, Laboratory of Algal Physiology, The University of Texas. These algal cultures had a density of 1.0 gm/L dry weight, or equivalent to 3.8 cmm/mL packed
cell volume, and had a chlorophyll content of 38 mg/L.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

25 °C

pH:

7

Nominal and measured concentrations:

control, 50, 100, 250, 500, 750, and 1000 mg/L (nominal)

Details on test conditions:

TEST SYSTEM
- Test vessel: Aquarium constructed of lucite; 48 inches long, 12 inches wide, 6.75 inches deep. To hold the test tubes which were used as the actual test vessels, two masonite boards with matched and equally spaced holes were positioned near the top and the bottom of the aquarium. A test tube contained 15 mL of inorganic culture medium with a predetermined amount of the tested organic compound and 5 mL of algal source culture suspension making a total volume of 20 mL. Covered with caps
- Aeration: A stream of 5% carbon dioxide in air gas mixture was supplied to provide the inorganic carbon souree and also to keep the algal cells in suspension
- No. of vessels per concentration (replicates): three test tubes during the screening tests, five tubes during the final testso

GROWTH MEDIUM
- Knop's solution, including the Hutner-EDTA microelement system


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Tap water

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: yes, with KOH
- Photoperiod: continuous
- Light intensity and quality: four 200-watt fluorescent lamps with attached aluminum reflectors; intensity of 550 to 650 foot-candles
- Temperature: Tap water with a constant head was continuously circulated through the aquarium to maintain a relatively constant temperature.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: A Beckman Spectrophotometer, Model DB, was used for all chlorophyll analysis. This spectrophotometer is a double beam instrument
for making transmittance and absorbance measurements in the 205 to 770 µm wavelength range
- Chlorophyll measurement: Measuring changes in chlorophyll content of the algal suspensions every 24 hours.
- Other: The Warburg manometric technique was used to measure the photosynthetic gas exchange that occurs when a carbonate-bicarbonate buffer is used as the suspending fluid

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

50 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: chlorophyll reduction

Validity criteria fulfilled:

not applicable

Reference 6

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

no guideline followed

Principles of method if other than guideline:

Two methods applied:
1) Algal cultures were grown in test tubes for a period of 72 hours. The toxicity was evaluated by measuring changes in chlorophyll content of the algal suspensions every 24 hours.
2) The Warburg manometric technique was used to measure the photosynthetic gas exchange that occurs when a carbonate-bicarbonate buffer is used as the suspending fluid.

GLP compliance:

no

Analytical monitoring:

no

Test organisms (species):

Chlorella pyrenoidosa

Details on test organisms:

An Emerson strain of bacteria-free, experimentally reproducible cultures of Chlorella pyrenoidosa was obtained from Dr. Jack E. Myers, Professor of Zoology and Botany and Director, Laboratory of Algal Physiology, The University of Texas. These algal cultures had a density of 1.0 gm/L dry weight, or equivalent to 3.8 cmm/mL packed
cell volume, and had a chlorophyll content of 38 mg/L.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

25 °C

pH:

7

Nominal and measured concentrations:

control, 50, 100, 250, 500, 750, and 1000 mg/L (nominal)

Details on test conditions:

TEST SYSTEM
- Test vessel: Aquarium constructed of lucite; 48 inches long, 12 inches wide, 6.75 inches deep. To hold the test tubes which were used as the actual test vessels, two masonite boards with matched and equally spaced holes were positioned near the top and the bottom of the aquarium. A test tube contained 15 mL of inorganic culture medium with a predetermined amount of the tested organic compound and 5 mL of algal source culture suspension making a total volume of 20 mL. Covered with caps
- Aeration: A stream of 5% carbon dioxide in air gas mixture was supplied to provide the inorganic carbon souree and also to keep the algal cells in suspension
- No. of vessels per concentration (replicates): three test tubes during the screening tests, five tubes during the final testso

GROWTH MEDIUM
- Knop's solution, including the Hutner-EDTA microelement system


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Tap water

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: yes, with KOH
- Photoperiod: continuous
- Light intensity and quality: four 200-watt fluorescent lamps with attached aluminum reflectors; intensity of 550 to 650 foot-candles
- Temperature: Tap water with a constant head was continuously circulated through the aquarium to maintain a relatively constant temperature.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: A Beckman Spectrophotometer, Model DB, was used for all chlorophyll analysis. This spectrophotometer is a double beam instrument
for making transmittance and absorbance measurements in the 205 to 770 µm wavelength range
- Chlorophyll measurement: Measuring changes in chlorophyll content of the algal suspensions every 24 hours.
- Other: The Warburg manometric technique was used to measure the photosynthetic gas exchange that occurs when a carbonate-bicarbonate buffer is used as the suspending fluid

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

50 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: chlorophyll reduction

Validity criteria fulfilled:

not applicable

Reference 7

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

static bioassay

GLP compliance:

not specified

Analytical monitoring:

no

Vehicle:

not specified

Test organisms (species):

other: Chlorella pyrenoidosa, Scenedesmus pannonicus, Selenastrum capricornutum

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Remarks on exposure duration:

C. pyrenoidosa and S. pannonicus: 48 h; S. capricornutum: 96 h

Test temperature:

C. pyrenoidosa and S. pannonicus: 25 °C
S. capricornutum: 26 °C

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

34 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Chlorella pyrenoidosa

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

36 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Scenedesmus pannonicus

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

65 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Selenastrum capricornutum

Validity criteria fulfilled:

not applicable

Reference 8

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

other: NEN-6506 (1980)

GLP compliance:

not specified

Analytical monitoring:

no

Vehicle:

not specified

Test organisms (species):

Selenastrum sp.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Test temperature:

26 ± 3 °C

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

75 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Validity criteria fulfilled:

not specified

Reference 9

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

no guideline followed

Principles of method if other than guideline:

Two methods applied:
1) Algal cultures were grown in test tubes for a period of 72 hours. The toxicity was evaluated by measuring changes in chlorophyll content of the algal suspensions every 24 hours.
2) The Warburg manometric technique was used to measure the photosynthetic gas exchange that occurs when a carbonate-bicarbonate buffer is used as the suspending fluid.

GLP compliance:

no

Analytical monitoring:

no

Test organisms (species):

Chlorella pyrenoidosa

Details on test organisms:

An Emerson strain of bacteria-free, experimentally reproducible cultures of Chlorella pyrenoidosa was obtained from Dr. Jack E. Myers, Professor of Zoology and Botany and Director, Laboratory of Algal Physiology, The University of Texas. These algal cultures had a density of 1.0 gm/L dry weight, or equivalent to 3.8 cmm/mL packed
cell volume, and had a chlorophyll content of 38 mg/L.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

25 °C

pH:

7

Nominal and measured concentrations:

control, 50, 100, 250, 500, 750, and 1000 mg/L (nominal)

Details on test conditions:

TEST SYSTEM
- Test vessel: Aquarium constructed of lucite; 48 inches long, 12 inches wide, 6.75 inches deep. To hold the test tubes which were used as the actual test vessels, two masonite boards with matched and equally spaced holes were positioned near the top and the bottom of the aquarium. A test tube contained 15 mL of inorganic culture medium with a predetermined amount of the tested organic compound and 5 mL of algal source culture suspension making a total volume of 20 mL. Covered with caps
- Aeration: A stream of 5% carbon dioxide in air gas mixture was supplied to provide the inorganic carbon souree and also to keep the algal cells in suspension
- No. of vessels per concentration (replicates): three test tubes during the screening tests, five tubes during the final testso

GROWTH MEDIUM
- Knop's solution, including the Hutner-EDTA microelement system


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Tap water

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: yes, with KOH
- Photoperiod: continuous
- Light intensity and quality: four 200-watt fluorescent lamps with attached aluminum reflectors; intensity of 550 to 650 foot-candles
- Temperature: Tap water with a constant head was continuously circulated through the aquarium to maintain a relatively constant temperature.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: A Beckman Spectrophotometer, Model DB, was used for all chlorophyll analysis. This spectrophotometer is a double beam instrument
for making transmittance and absorbance measurements in the 205 to 770 µm wavelength range
- Chlorophyll measurement: Measuring changes in chlorophyll content of the algal suspensions every 24 hours.
- Other: The Warburg manometric technique was used to measure the photosynthetic gas exchange that occurs when a carbonate-bicarbonate buffer is used as the suspending fluid

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

50 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: chlorophyll reduction

Validity criteria fulfilled:

not applicable

Reference 10

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

no guideline followed

Principles of method if other than guideline:

Two methods applied:
1) Algal cultures were grown in test tubes for a period of 72 hours. The toxicity was evaluated by measuring changes in chlorophyll content of the algal suspensions every 24 hours.
2) The Warburg manometric technique was used to measure the photosynthetic gas exchange that occurs when a carbonate-bicarbonate buffer is used as the suspending fluid.

GLP compliance:

no

Analytical monitoring:

no

Test organisms (species):

Chlorella pyrenoidosa

Details on test organisms:

An Emerson strain of bacteria-free, experimentally reproducible cultures of Chlorella pyrenoidosa was obtained from Dr. Jack E. Myers, Professor of Zoology and Botany and Director, Laboratory of Algal Physiology, The University of Texas. These algal cultures had a density of 1.0 gm/L dry weight, or equivalent to 3.8 cmm/mL packed
cell volume, and had a chlorophyll content of 38 mg/L.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

25 °C

pH:

7

Nominal and measured concentrations:

control, 50, 100, 250, 500, 750, and 1000 mg/L (nominal)

Details on test conditions:

TEST SYSTEM
- Test vessel: Aquarium constructed of lucite; 48 inches long, 12 inches wide, 6.75 inches deep. To hold the test tubes which were used as the actual test vessels, two masonite boards with matched and equally spaced holes were positioned near the top and the bottom of the aquarium. A test tube contained 15 mL of inorganic culture medium with a predetermined amount of the tested organic compound and 5 mL of algal source culture suspension making a total volume of 20 mL. Covered with caps
- Aeration: A stream of 5% carbon dioxide in air gas mixture was supplied to provide the inorganic carbon souree and also to keep the algal cells in suspension
- No. of vessels per concentration (replicates): three test tubes during the screening tests, five tubes during the final testso

GROWTH MEDIUM
- Knop's solution, including the Hutner-EDTA microelement system


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Tap water

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: yes, with KOH
- Photoperiod: continuous
- Light intensity and quality: four 200-watt fluorescent lamps with attached aluminum reflectors; intensity of 550 to 650 foot-candles
- Temperature: Tap water with a constant head was continuously circulated through the aquarium to maintain a relatively constant temperature.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: A Beckman Spectrophotometer, Model DB, was used for all chlorophyll analysis. This spectrophotometer is a double beam instrument
for making transmittance and absorbance measurements in the 205 to 770 µm wavelength range
- Chlorophyll measurement: Measuring changes in chlorophyll content of the algal suspensions every 24 hours.
- Other: The Warburg manometric technique was used to measure the photosynthetic gas exchange that occurs when a carbonate-bicarbonate buffer is used as the suspending fluid

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

50 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: chlorophyll reduction

Validity criteria fulfilled:

not applicable

Reference 11

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

18 October 2004 to 21 October 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

yes

Remarks:

see remarks below under "Principles of method if other than guideline"

Principles of method if other than guideline:

Deviations: The test temperature was outside the specifed temperature range, at 22 - 23°C between the 0 and 24 h and the 24 and 48 h observation intervals. The photosynthetically-active radiation (PAR) was not measured at test initiation due to unavailability of the meter. The PAR was measured 7 days after test initiation in lieu. These deviations were not considered to adversely affect the integrity of the study.

GLP compliance:

yes

Analytical monitoring:

not required

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A 300 mg/mL stock solution was prepared by placing 7.4 mL of ethyl phenols in a 25 mL volumetric flask and diluting to volume with acetone.
- Controls: untreated algal medium only for untreated control and acetone vehicle control (AAP medium plus 300 mg/L sodium bicarbonate)
- Chemical name of vehicle (organic solvent, emulsifier or dispersant):
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): Stock solution concentrations: 300, 250, 130, 63, 31 and 16 mg/mL. Final test solution concentrations: 25, 13, 6.3, 3.1 and 1.6 mg/L.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): none

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: 1648
- Source (laboratory, culture collection): Universtiy of Texas, Austin, Texas, USA
- Age of inoculum (at test initiation): 3 days
- Method of cultivation: Continuous agitation on an orbital shaker (100 rpm) in 125 mL glass flasks each containing 50 mL of medium, covered with stainless steel caps permitting gas exchange.

ACCLIMATION
- Acclimation period: 3 days
- Culturing media and conditions (same as test or not): yes, except screw cap tops used during test.
- Any deformed or abnormal cells observed: not stated

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

22 - 24 °C

pH:

8.2 - 9.9

Nominal and measured concentrations:

Control, acetone control, 1.6, 3.1, 6.3, 13 and 25 mg/L (nominal)
Control, acetone control, 1.1, 2.0, 5.2, 16 and 22 mg/L (mean measured)

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 250 mL glass Erlenmeyer flasks with screw cap tops, 100 mL per flask.
- Aeration: none
- Initial cells density: approx. 1.0 x 10E4 cells/mL
- Control end cells density:
- No. of organisms per vessel:
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3
- No. of vessels per vehicle control (replicates): 3

GROWTH MEDIUM
- Standard medium used: yes
- Detailed composition if non-standard medium was used: not applicable

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: sterile de-ionised water
- Total organic carbon: 1.7 mg/L
- Particulate matter: not observed
- Metals: not present in toxic concentrations
- Pesticides: not present in toxic concentrations
- Chlorine: not stated
- Alkalinity: not stated
- Ca/mg ratio: not stated
- Conductivity: 350 - 360 µmhos/cm at test initiation
- Culture medium different from test medium: no
- Intervals of water quality measurement: 0 and 72 h

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: continuous light
- Light intensity and quality: 7000 to 9100 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter]
- Chlorophyll measurement: not stated

TEST CONCENTRATIONS
- Spacing factor for test concentrations: control, acetone control, 1.6, 3.1, 6.3, 13 and 25 mg/L (nominal) for definitive test
- Range finding study
- Test concentrations: control, solvent control, 0.010, 0.10, 1.0 and 10 mg/L
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

no

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

5.2 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% CL

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

17 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95 % CL (14-19 mg/L)

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 22 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

5.2 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Analytical analysis of the test media showed the mean measured exposure concentration ranged between 65 - 120% of nominal, i.e. 1.1, 2.0, 5.2, 16 and 22 mg/L. At the end of the exposure period, the analytical result from the 6.3 mg/L nominal dose without algae present was 4.8 mg/L, and from the same dose with algae present the result was 4.2 mg/L, demonstrating that the presence of algae had little impact on the concentration of ethyl phenols. Percentage recoveries from the six QC samples demonstrated consistency with the predetermined recovery range (57.4 - 68.6% for the 1.00 mg/L samples and 88.7 - 99.9% for 10.0 and 50.0 mg/L samples).

Cell densities are presented in Table 2. By the end of the test, cells exposed to all treatment levels were observed to be normal. Mean cell densities in the control and solvent control, respectively were 144.56 and 166.78 x 10E4 cells/mL. Mean cell densities in the 1.1, 2.0, 5.2, 16 and 22 mg/L treatments were 195.50, 152.78, 139.06, 81.50 and 56.50 x 10E4 cells/mL, respectively. A significant reduction in total biomass was observed in the 16 and 22 mg/L treatments compared to the pooled control data.

Mean growth rates were 1.62 and 1.66 dayE-1 for the control and acetone control after 72 hours, respectively. There were no significant differences between the control and solvent control growth rates so these values were pooled. The mean 0 to 72 hours growth rate at 1.1, 2.0, 5.2, 16 and 22 mg/L was 1.71, 1.64, 1.60, 1.43 and 1.31 daysE-1, respectively. A significant reduction in growth was observed in the 16 and 22 mg/L treatments compared to the pooled control data.

Table 1: Concentrations of ethyl phenols measured in the exposure solutions during the 72 hour exposure to Pseudokirchneriella subcapitata

Nominal concentration (mg/L)	Measured concentration (mg/L)
	0 hour	72 hour	mean	% of nominal
Control	<0.14	<0.14	NA	NA
Solvent control^a	<0.14	<0.14	NA	NA
1.6	1.3	0.86	1.1	69
3.1	2.5	1.6	2.0	65
6.3	6.1	4.2/4.8^b	5.2	82
13	19	13	16	120
25	22	22	22	89

NA = not applicable
^a= acetone added at a concentration of 0.1 mL/L
^b= result of the additional sample without algae present to determine biological uptake/degradation

 Table 2: Cell density of Pseudokirchneriella subcapitata after exposure to ethyl phenols

Mean measured concentration (mg/L)	Cell density (x 10^4cells/mL)
	Observation interval (hours)
	Replicate	24	48	72
Control	A	5.75	22.75	148.00
	B	6.25	34.50	132.00
	C	5.50	47.25	153.67
	Mean (SD)	5.83 (0.38)	34.83 (12.25)	144.56 (11.24)
Solvent control	A	5.25	32.00	196.00
	B	8.25	27.75	157.67
	C	6.50	26.50	146.67
	Mean (SD)	6.67 (1.51)	28.75 (2.88)	166.78 (25.9)
1.1	A	7.50	24.25	174.00
	B	8.25	15.00	260.50
	C	4.50	27.25	152.00
	Mean (SD)	6.75 (1.98)	22.17 (6.39)	195.50 (57.36)
2.0	A	6.25	27.75	163.67
	B	6.00	28.25	150.67
	C	7.50	20.00	144.00
	Mean (SD)	6.58 (0.8)	25.33 (4.63)	152.78 (10)
5.2	A	5.25	27.00	123.75
	B	6.00	30.50	187.67
	C	6.00	21.25	105.75
	Mean (SD)	5.75 ()0.43	26.25 (4.67)	139.06 (43.05)
16	A	4.75	6.75	82.50
	B	5.00	22.75	90.00
	C	6.25	29.00	72.00
	Mean (SD)	5.33 (0.8)	19.50 (11.48)	81.50 (9.04)
22	A	3.75	9.75	64.50
	B	5.50	13.00	60.75
	C	2.75	15.50	44.25
	Mean (SD)	4.00 (1.39)	12.75 (2.88)	56.50 (10.77)

SD = standard deviation

 

Table 3: Calculated biomass (area under the growth curve) of Pseudokirchneriella subcapitata after exposure to ethyl phenols

Mean measured concentration (mg/L)	Biomass (x 10^4cells.days/mL)
	Observation interval (hours)
	Replicate	0-24	24-48	48-72	72 hour total area	% inhibition^a
Control	A	2.57	12.65	87.01	102.24	-
	B	2.84	18.50	84.82	106.16	-
	C	2.44	24.23	102.57	129.23	-
	Mean (SD)	2.62 (0.21)	18.46 (5.79)	91.47 (9.68)	112.54 (14.59)	NA
Solvent control	A	2.30	16.83	116.53	135.66	-
	B	3.93	16.23	94.57	114.73	-
	C	2.98	14.80	88.26	106.04	-
	Mean (SD)	3.07 (0.82)	15.95 (1.04)	99.79 (14.84)	118.81 (15.23)	NA
Pooled control	Mean (SD)	-	-	-	115.68 (13.77)	NA
1.1	A	3.52	14.20	101.19	118.92	-
	B	3.93	10.15	141.02	155.10	-
	C	1.90	14.20	91.39	107.49	-
	Mean (SD)	3.11 (1.07)	12.85 (2.34)	111.20 (26.29)	127.17 (24.85)	-10
2.0	A	2.84	15.28	97.67	115.79	-
	B	2.71	15.40	91.22	109.33	-
	C	3.52	12.17	83.53	99.23	-
	Mean (SD)	3.02 (0.44)	14.28 (1.83)	90.81 (7.08)	108.11 (8.35)	7
5.2	A	2.30	14.44	76.70	93.44	-
	B	2.71	16.47	111.46	130.64	-
	C	2.71	12.06	64.45	79.22	-
	Mean (SD)	2.57 (0.23)	14.32 (2.21)	84.2 (24.39)	101.10 (26.55)	13
16	A	2.03	4.54	44.99	51.56	-
	B	2.17	12.29	57.11	71.57	-
	C	2.84	15.87	51.05	69.77	-
	Mean (SD)	2.35 (0.44)	10.90 (5.8)	51.05 (6.06)	64.30 (11.07)^b	44
22	A	1.49	5.49	37.25	44.23	-
	B	2.44	7.88	37.00	47.31	-
	C	0.95	7.76	29.78	38.48	-
	Mean (SD)	1.63 (0.75)	7.04 (1.35)	34.68 (4.24)	43.34 (4.48)	63

SD = standard deviation
NA = not applicable
^a= % inhibition relative to the pooled control
^b= Significantly reduced as compared to the pooled control, based on William’s Test (p<0.05)

Table 4: Calculated growth rates of Pseudokirchneriella subcapitata after exposure to ethyl phenols

Mean measured concentration (mg/L)	Growth rate (days^-1)
	Observation interval (hours)
	Replicate	24	48	72	% inhibition^a
Control	A	1.61	1.53	1.63	-
	B	1.69	1.74	1.59	-
	C	1.57	1.89	1.64	-
	Mean (SD)	1.63 (0.06)	1.72 (0.18)	1.62 (0.03)	NA
Solvent control	A	1.53	1.70	1.72	-
	B	1.95	1.63	1.65	-
	C	1.73	1.61	1.63	-
	Mean (SD)	1.74 (0.21)	1.65 (0.05)	1.66 (0.05)	NA
Pooled control	Mean (SD)	-	-	1.64 (0.04)	NA
1.1	A	1.86	1.56	1.68	-
	B	1.95	1.33	1.81	-
	C	1.39	1.62	1.64	-
	Mean (SD)	1.73 (0.3)	1.50 (0.16)	1.71 (0.09)	-4
2.0	A	1.69	1.63	1.66	-
	B	1.65	1.64	1.63	-
	C	1.86	1.47	1.62	-
	Mean (SD)	1.74 (0.11)	1.58 (0.1)	1.64 (0.02)	0
5.2	A	1.53	1.62	1.57	-
	B	1.65	1.68	1.71	-
	C	1.65	1.50	1.52	-
	Mean (SD)	1.61 (0.07)	1.60 (0.09)	1.60 (0.10)	2
16	A	1.44	0.94	1.44	-
	B	1.49	1.53	1.47	-
	C	1.69	1.65	1.39	-
	Mean (SD)	1.54 (0.13)	1.37 (0.38)	1.43 (0.04)^b	13
22	A	1.22	1.12	1.36	-
	B	1.57	1.26	1.34	-
	C	0.93	1.34	1.23	-
	Mean (SD)	1.24 (0.32)	1.24 (0.11)	1.31 (0.07)^b	20

SD = standard deviation
NA = not applicable
^a= % inhibition relative to the pooled control
^b= Significantly reduced as compared to the pooled control, based on William’s Test (p<0.05)

Validity criteria fulfilled:

yes

Conclusions:

The 72 hour EC50 for total biomass was determined to be 17 mg/L and the NOEC was 5.2 mg/L.
The 72 hour EC50 for growth rate was determined to be >22 mg/L and the NOEC was 5.2 mg/L.

Reference 12

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

yes

Remarks:

The temperature in the test solutions deviated from 24 ± 1 °C and was instead in the range 22 - 23°C between teh 48 and 72 hour observaton periods. This was not considered to adversely impact the integrity of the study.

GLP compliance:

yes

Analytical monitoring:

yes

Vehicle:

yes

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: 1648
- Source (laboratory, culture collection): Universtiy of Texas, Austin, Texas, USA
- Age of inoculum (at test initiation): 3 days
- Method of cultivation: Continuous agitation on an orbital shaker (100 rpm) in 125 mL glass flasks each containing 50 mL of medium, covered with stainless steel caps permitting gas exchange.

ACCLIMATION
- Acclimation period: 3 days
- Culturing media and conditions (same as test or not): yes, except screw cap tops used during test.
- Any deformed or abnormal cells observed: none stated

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

22-24°C

pH:

pH 8.2-8.4 (test start), pH 8.9-9.5 (test end)

Nominal and measured concentrations:

Nominal: 1.6, 3.1, 6.3, 13 and 25 mg/L
Measured: 1.0, 1.7, 4.4, 10 and 22 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 250 mL glass Erlenmeyer flasks with screw cap tops, 100 mL per flask.
- Aeration: none
- Type of flow-through (e.g. peristaltic or proportional diluter): static test
- Renewal rate of test solution (frequency/flow rate): closed system
- Initial cells density: approx. 1.0 x 10E4 cells/mL
- Control end cells density:
- No. of organisms per vessel:
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3
- No. of vessels per vehicle control (replicates): 3

GROWTH MEDIUM
- Standard medium used: yes
- Detailed composition if non-standard medium was used: not applicable

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: sterile de-ionised water
- Total organic carbon: 1.7 mg/L
- Particulate matter: not observed
- Metals: not present in toxic concentrations
- Pesticides: not present in toxic concentrations
- Chlorine: not stated
- Alkalinity: not stated
- Ca/mg ratio: not stated
- Conductivity: 350 - 380 µmhos/cm
- Culture medium different from test medium:
- Intervals of water quality measurement: 0 and 72 h

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: continuous light
- Light intensity and quality: 7000 - 8600 lux, photosynthetically active radiation (PAR) was 95 - 127 µE/m2/s
- Salinity (for marine algae): not applicable

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter]
- Chlorophyll measurement: not stated

TEST CONCENTRATIONS
- Spacing factor for test concentrations: approx. 2
- Justification for using less concentrations than requested by guideline: not applicable
- Range finding study
- Test concentrations: control, solvent control, 0.010, 0.10, 1.0 and 10 mg/L
- Results used to determine the conditions for the definitive study: yes

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

14 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95 % CL: 12-15 mg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.7 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 22 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.7 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes/no
- Observation of abnormalities (for algal test):
- Unusual cell shape: In the 10 mg/L exposure solution, cells were observed to be bloated at 48 hours. In the 10 and 22 mg/L exposure solutions, at 72 hours cells were observed to be bloated and cell fragments were seen.
- Colour differences: not stated
- Flocculation: not stated
- Adherence to test vessels: not stated
- Aggregation of algal cells: not stated
- Other:
- Any stimulation of growth found in any treatment: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: none stated
- Effect concentrations exceeding solubility of substance in test medium: no

Analytical analysis of the test media showed the mean measured exposure concentration ranged between 53 - 93% of nominal, i.e. 1.0, 1.7, 4.4, 10 and 22 mg/L. At

the end of the exposure period, the analytical result from the 6.3 mg/L nominal dose without algae present was 4.6 mg/L. From the 6.3 mg/L test solution with algae present, the measured concentration was 3.3 mg/L. This demonstrates that the presence of algae had a slight impact on the concentration of mixed xylenols in the test solution. Percentage recoveries in teh six QC samples demonstrated consistency with teh predetermined recovery range (60.4 - 65.2% for the 1.00 mg/L samples and 87.4 - 100% for the 10.0 and 50.0 mg/L samples).

Cell densities were determined at each observation interval. In the 10 mg/L exposure solution, cells were observed to be bloated at 48 hours. In the 10 and 22 mg/L exposure solutions, at 72 hours cells were observed to be bloated and cell fragments were seen. Cells exposed to 1.0, 1.7 and 4.4 mg/L were observed to be normal. The 72 hour cell density in the control and solvent control had a mean of 32.67 and 83.25 x 10E4 cells/mL, respectively. Cell densities in the 1.0, 1.7, 4.4, 10 and 22 mg/L treatment levels had a mean of 82.67, 83.00, 72.1, 53.00 and 13.92 x 10E4 cells/mL, respectively. A significant difference was observed in the total biomass between the control and solvent control (t-test) and so solvent control data were used to determine the treatment level effects. A significant reduction in total biomass was observed in the 4.4, 10 and 22 mg/L treatment levels compared to the solvent control data, using Williams' test.

Mean growth rates were 1.17 and 1.49 daysE-1 for teh control and solvent control, respectively after 72 hours. The t-test determined a significant difference between teh control and solvent control growth rate and so solvent control data were used to determine treatment related effects. The mean growth rates in the 1.0, 1.7, 4.4, 10 and 22 mg/L treatments were 1.49, 1.49, 1.44, 1.34 and 0.89 daysE-1, respectively. These data showed a significant reduction in growth rate with Williams' test for teh 4.4, 10 and 22 mg/L treatment levels, compared to the solvent control data.

Table 1: Concentrations of mixed xylenols measured during the 72 hour exposure of Pseudokirchneriella subcapitata

Nominal concentration (mg/L)	Measured concentration (mg/L)
	0 hour	72 hours	Mean	% of nominal
Control	<0.14	<0.14	NA	NA
Solvent control	<0.14	<0.14	NA	NA
1.6	1.3	0.70	1.0	63
3.1	2.2	1.1	1.7	53
6.3	5.5	3.3/4.6	4.4	69
13	11	9.4	10	79
25	24	21	22	90

 

Table 2: Cell density of Pseudokirchneriella subcapitata after 24, 48 and 72 hours of exposure to mixed xylenols

Mean measured concentration (mg/L)	Cell density (x 10^4cells/mL)
	Observation interval (hours)
		24	48	72
Control	A	9.50	35.75	33.50
	B	3.50	26.50	35.25
	C	4.00	20.25	29.25
	Mean (SD)	5.67 (3.33)	27.50 (7.8)	32.67 (3.09)
Solvent control	A	2.75	25.25	85.00
	B	6.25	27.25	84.25
	C	5.25	34.75	80.50
	Mean (SD)	4.75 (1.8)	29.08 (5.01)	83.25 (2.41)
1.0	A	6.00	26.50	75.75
	B	6.00	32.75	81.25
	C	6.00	19.75	91.00
	Mean (SD)	6.00 (0)	26.33 (6.5)	82.67 (7.72)
1.7	A	4.25	28.25	84.00
	B	4.25	32.25	85.75
	C	6.25	28.25	79.25
	Mean (SD)	4.92 (1.15)	29.58 (2.31)	83.00 (3.36)
4.4	A	3.75	25.50	70.00
	B	4.50	21.50	78.25
	C	6.25	21.25	68.25
	Mean (SD)	4.83 (1.28)	22.75 (2.38)	72.17 (5.34)
10	A	3.50	15.00	51.50
	B	6.25	18.75	57.75
	C	4.75	17.75	49.75
	Mean (SD)	4.83 (1.38)	17.17 (1.94)	53.00 (4.21)^ab
22	A	1.25	8.00	15.75
	B	1.50	8.50	12.25
	C	3.00	6.75	13.75
	Mean (SD)	1.92 (0.95)	7.75 (0.9)^a	13.92 (1.76)^ab

SD = Standard deviation.
^a= Cells were observed to be bloated.
^b= Cell fragments were observed.

 

Table 3: Calculated biomass (area under the growth curve) of Pseudokirchneriella subcapitata after 24, 48 and 72 hours of exposure to mixed xylenols

Mean measured concentration (mg/L)	Biomass (x 10^4cells.days/mL)
	Observation interval (hours)
		0-24	24-48	48-72	72 hour total area	% inhibition^a
Control	A	4.52	23.20	28.02	55.74	-
	B	1.33	15.02	24.90	41.24	-
	C	1.59	11.94	19.79	33.32	-
	Mean (SD)	2.48 (1.77)	16.72 (5.82)	24.24 (4.15)	43.43 (11.37)	NA
Solvent control	A	0.93	13.95	45.10	59.98	-
	B	2.79	16.90	45.62	65.31	-
	C	2.26	20.39	47.19	69.83	-
	Mean (SD)	1.99 (0.96)	17.08 (3.22)	45.97 (1.08)	65.04 (4.93)	NA
1.0	A	2.66	16.36	41.77	60.79	-
	B	2.66	19.71	46.67	69.04	-
	C	2.66	12.74	45.31	60.71	-
	Mean (SD)	2.66 (0)	16.27 (3.49)	44.58 (2.53)	63.51 (4.79)	2
1.7	A	1.73	16.36	45.94	64.03	-
	B	1.73	18.51	48.33	68.57	-
	C	2.79	17.43	43.96	64.18	-
	Mean (SD)	2.08 (0.61)	17.43 (1.07)	46.08 (2.19)	65.59 (2.58)	-1
4.4	A	1.46	14.62	38.96	55.04	-
	B	1.86	12.88	40.73	55.46	-
	C	2.79	13.68	36.46	52.93	-
	Mean (SD)	2.04 (0.68)	13.72 (0.87)	38.72 (2.15)	54.48 (1.36)^b	16
10	A	1.33	8.85	26.87	37.05	-
	B	2.79	12.34	31.04	46.17	-
	C	1.99	11.00	27.29	40.28	-
	Mean (SD)	2.04 (0.73)	10.73 (1.76)	28.40 (2.29)	41.17 (4.62)	37
22	A	0.13	3.89	9.06	13.08	-
	B	0.27	4.29	7.81	12.37	-
	C	1.06	4.16	7.71	12.93	-
	Mean (SD)	0.49 (0.50)	4.11 (0.20)	8.19 (0.75)	12.79 (0.38)^b	80

SD = Standard deviation
NA = Not applicable
^a= % inhibition relative to the solvent control.
^b= Significantly reduced compared to the solvent control, based on Williams’ test.

Validity criteria fulfilled:

yes

Conclusions:

The 72 hour EC50 for total biomass was determined to be 14 mg/L and the NOEC was 1.7 mg/L.
The 72 hour EC50 for growth rate was determined to be >22 mg/L and the NOEC was 1.7 mg/L.

Reference 13

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

04 Mar - 06 Apr 2019

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:

adopted March 2006, corrected July 28, 2011

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OECD Series on Testing and Assessment, No. 23

Version / remarks:

February 08, 2019

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: SANCO/3029/99 rev.4 11/07/00

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Hessisches Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Control and all test item concentrations
- Sampling method: Duplicate samples were taken at test start and test end (72 h, by pouring together the contents of the test beakers of each treatment).
- Sample storage conditions before analysis: All samples were stored in a refrigerator (4 ± 4 °C).

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test medium of the highest test concentration of nominal 80 mg test item/L was prepared by dissolving 84 mg test item into 1050 mL test water by intense stirring for 15 min. Adequate volumes of this test medium were diluted with test water to prepare the test media of the other desired test concentrations.
- Controls: Medium without test substance

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Green algae
- Strain: 61.81 SAG
- Source (laboratory, culture collection): Sammlung von Algenkulturen, Albrecht-von-Haller-Institut für Pflanzen-wissenschaften, Universität Göttingen, Göttingen, Germany
- Age of inoculum (at test initiation): 3 days
- Method of cultivation: The algae were cultivated in the laboratories of the test facility under standardised conditions according to the test guidelines.

ACCLIMATION
- Acclimation period: 3 days
- Culturing media and conditions (same as test or not): same as test

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

22.3 - 22.7

pH:

8.1 - 9-.5 (Control)
8.2 - 9.5 (Test Item Concentrations)

Nominal and measured concentrations:

Control, 0.238, 0.763, 2.44, 7.81, 25 and 80 mg test item/L

Details on test conditions:

TEST SYSTEM
Test vessel:
- Type: closed
- Material, size, headspace, fill volume: 50 mL-Erlenmeyer flasks, containing as much test medium as possible, closed with a conical glass stopper
- Initial cells density: 5000 algal cells per mL
- Control end cells density: 83.339 x 10E+04 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes (media was enriched with 300 mg NaHCO3/L to enable algae growth in closed vessels)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Analytical grade salts were added
- Hardness: 0.24 mmol/L (=24 mg/L)
- Culture medium different from test medium: No
- Intervals of water quality measurement: daily

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Photoperiod: Continuous illumination
- Light intensity and quality: 6215 lux (range: 5660 to 6530 lux)

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: Cell density was determined by spectrophotometric measurement

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.16
- Range finding study: a non-GLP range-finding test was performed

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

27.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidential interval

Remarks:

22.4 - 34.3

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

5.92 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % conf. interval

Remarks:

2.94 - 8.85

Details on results:

- Exponential growth in the control: yes
- Observation of abnormalities (for algal test): The microscopic examination of the shape of the algal cells after 72 h of test duration did show differences between the algae that had been growing up at all test item treated groups and the algal cells in the control. The algal cells in the test item treated groups were bigger and showed deformations (were more oval and appeared clumsy).

Results with reference substance (positive control):

Results of the Most Recent Test with Pseudokirchneriella subcapitata performed with the Reference Item Potassium Dichromate in June 2018 (Study No.: 129862210): EC50 (72 h) 1.11 mg/L

Reported statistics and error estimates:

Based on the calculated cell densities, the 72 h ErC50 and the corresponding EC20 and EC10 values and where possible their 95% confidence limits were calculated by Weibull analysis. The 72 h EyC50 and the corresponding EC20 and EC10 values and where possible their 95% confidence limits were calculated by Probit analysis with weighted linear regression. For the determination of the 72 h LOEC and the 72 h NOEC, the calculated growth rates and yields at each test concentration were tested for significant differences compared to the control values by Williams t-test. The software used to perform the statistical analysis was ToxRat Professional, Version 3.2.1, ToxRat Solutions GmbH.

Analytical Results:

Table 1: Summary of Analytical Results

Sample Description	Total Organic Carbon
nominal concentration	TOC	RSD
[mg test item/L]	[mg Carbon/L]1	[%]	n
Control	n.a.	n.a.	2
0.238	<LOQ	n.a.	4
0.763	<LOQ	n.a.	4
2.44	<LOQ	n.a.	4
7.81	5.42	14	4
25	17.67	9	4
80	63.54	1	4

1 mean value of all measured samples per treatment group
RSD: relative standard deviation per treatment group
n: number of analysed samples; n.a.: not applicable

Biological Results:

Table 2: Growth rates µ and Percentage Inhibition of µ during the test period

Nominal concentration	Growth rates µ [1/day] and % inhibition of µ
	0–24 h		0-48 h		0-72 h
[mg test item/L]	µ	%	µ	%	µ	%
Control	1.581	-	1.816	-	1.705	-
0.238	1.710	-8.2	1.837	-1.1	1.696	0.5
0.763	1.618	-2.4	1.806	0.6	1.528	10.4
2.44	1.752	-10.8	1.749	3.7	1.528	11.9
7.81	1.750	-10.7	1.690	6.9	1.357	20.4
25	1.134	28.3	1.094	39.8	1.079	36.7
80	0.169	89.3	0.423	76.7	0.000	1000.0

 

Validity criteria for OECD 201.

Criterion from the guideline	Outcome	Validity criterion fulfilled
The biomass in the control cultures should have increased exponentially by a factor of at least 16 within the 72-hour test period.	166.7	Yes
The mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3, for 72-hour tests) in the control cultures must not exceed 35%	18.0%	Yes
The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 7% in tests with Pseudokirchneriella subcapitata and Desmodesmus subspicatus. For other less frequently tested species, the value should not exceed 10%.	0.9%	Yes

Validity criteria fulfilled:

yes

Remarks:

For further details please refer to “Any other information on results incl. tables”.

Description of key information

Based on all available information (weight-of-evidence) and following an analogue read-across and worst case approach the EC50 (48h) of 7.8 mg/L determined with Desmodesmus subspicatus (Kühn & Pattard, 1990) for p-cresol and the NOEC (72h) of 1.7 mg/L determined with Pseudokirchneriella subcapitata (Hoberg, 2005a) for mixed xylenols represent the most sensitive effect values for acute and chronic algae toxicity for constituents of Tar acids, Xylenol fraction (CAS 84989-06-0) and are considered key values for the chemical safety assessment. This values are lower than the effects determined with Pseudokirchneriella subcapitata for the UVCB mixture Tar acids, Xylenol fraction (CAS 84989-06-0) as a whole (72h-ErC50 = 27.9 mg/L and 72h-ErC10 = 5.92 mg/L) (Lührs & Schneider, 2019).

Key value for chemical safety assessment

EC50 for freshwater algae:

7.8 mg/L

EC10 or NOEC for freshwater algae:

1.7 mg/L

Additional information

One experimental study is available investigating the algae toxicity of Tar acids, Xylenol fraction (CAS 84989-06-0) to the unicellular green algae Pseudokirchneriella subcapitata (Lührs & Schneider, 2019). The study was performed according to OECD guideline 201 and GLP. The algae were exposed to nominal test item concentrations of 0.238, 0.763, 2.44, 7.81, 25 and 80 mg test item/L. The exposure concentrations during the test were verified by total organic carbon measurements. The analysed concentrations of the carbon content of the test item were within ± 20% of the measured initial concentrations during the test. The 72h-ErC50 was calculated to be 27.9 mg/L and the 72h-ErC10 5.92 mg/L.

Numerous further data on the toxicity to aquatic algae are available for constituents of Tar acids, Xylenol fraction (CAS 84989-06-0). These data are used to determine the hazard of Tar acids, Xylenol fraction (CAS 84989-06-0) by applying a weight of evidence and an analogue read-across approach. The studies were conducted with different species of freshwater algae. From the table below it can be seen that available data demonstrate effect values for acute and chronic to aquatic algae in a range from 7.8 to 100 mg/L and from 1.7 to 65 mg/L, respectively. The most sensitive acute value (Desmodesmus subspicatus) was determined in the study by Kühn & Pattard, (1990) with an EC50 (48h) of 7.8 mg/L for p-cresol. The most sensitive chronic effect value is a NOEC (72h) of 1.7 mg/L determined for mixed xylenols in a test with Pseudokirchneriella subcapitata (Hoberg, 2005a). These values are considered for PNEC derivation and risk assessment and for deriving environmental classification. For details on the read-across approach please refer to the analogue justification provided in IUCLID section 13.

Acute and chronic effect value for algae growth inhibition determined for Tar acids, Xylenol fraction:

Source	*	Species
Lührs & Schneider, 2019	f	Pseudokirchneriella subcapitata	ErC50 (72h) = 27.9 mg/L ErC10 (72h) = 5.92 mg/L

Acute and chronic effect values determined in the numerous studies on constituents of Tar acids, Xylenol fraction:

Source	*	Species	p-cresol	m-cresol	o-cresol
Slooff et al., 1983	f	Chlorella pyrenoidosa	-	-	NOEC (48h) = 34 mg/L
Slooff et al., 1983	f	Scenedesmus pannonicus	-	-	NOEC (48h) = 36 mg/L
Slooff et al., 1983	f	Selenastrum capricornutum (=Pseudokirchneriella subcapitata)	-	-	NOEC (48h) = 65 mg/L
Slooff, 1982	f	Selenastrum sp.	-	-	EC50 (48h) = 75 mg/L EC50 (96h) = 100 mg/L
Bringmann et al., 1975-1978	f	Microcystis aeruginosa	-	TGK** (8d) = 13 mg/L	TGK** (8d) = 6.8 mg/L
Kühn & Pattard, 1990	f	Desmodesmus subspicatus	EC50 (48h) = 7.8 mg/L EC10 (48h) = 4.6 mg/L	-	-
			2,4-xylenol	2,5-xylenol	2,6-xylenol
Huang & Gloyna, 1967	f	Chlorella pyrenoidosa	NOEC (72h) = 50 mg/L	NOEC (72h) = 50 mg/L	NOEC (72h) = 50 mg/L
			3,4-xylenol	3,5-xylenol
Huang & Gloyna, 1967	f	Chlorella pyrenoidosa	NOEC (72h) = 50 mg/L	NOEC (72h) = 50 mg/L	-
			Mixed xylenols	Mixed ethylphenols
Hoberg, 2005a	f	Pseudokirchneriella subcapitata	EC50 (72h) > 22 mg/L NOEC (72h) = 1.7 mg/L	-	-
Hoberg, 2005b	f	Pseudokirchneriella subcapitata	-	EC50 (72h) > 22 mg/L NOEC (72h) = 5.2 mg/L	-

* f = freshwater; ** TGK = Toxicity threshold, determined at 1% effect compared to control