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Toxicity to aquatic algae and cyanobacteria

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Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25 November 1997 - 29 November 1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to EC, OECD, and US EPA test guidelines, and in compliance with GLP.
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OTS 797.1050 (Algal Toxicity, Tiers I and II)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Concentrations Nominal loadings: 0 (control), 0.46, 1, 2.2, 4.6, 10, 22, 46, and 100 mg/L
- Sampling method: Duplicate samples (100 mL) were taken from control and test cultures at 0 hours and at 96 hours (replicates pooled) for analysis. All media were filtered prior to analysis.
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Method followed advice given in ECETOC 1996 Monograph No. 26 as the test material has poor water solubility. A stock solution of test material in chloroform (1000 mg/mL) was prepared, then further stock solutions diluted using chloroform as required to give stock solutions at 1000, 460, 220, 100, 46, 22, 10 and 4.6 mg/mL. 200 µL aliquots of each stock solution were added to autoclaved 2L vessels and the solvent was allowed to evaporate. 2 litres of algal medium were added to each vessel, then stirred for at least 18 hours using a magnetic stirrer with a 10 cm magnetic follower stirring at approximately 250 revolutions/minute. After stirring, the solutions were allowed to settle for approximately 1 hour, then a 500 mL aliquot of the water accomodated fraction was removed via a side tap from a mid water position.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): None reported.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Unicellular green algae
- Strain: CCAP 278/4
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa, Institute of Freshwater Ecology, Cumbria, UK.
- Method of cultivation: Sterile nutrient medium was inoculated from a master culture and cultured under continuous illumination (c. 7000 lux) in an orbital incubator at 22°C to give an algal suspension in log phase growth, characterised by a cell density of 1.4E+07 cells / ml.

ACCLIMATION
- Acclimation period:
- Culturing media and conditions (same as test or not): Lighting conditions used were equivalent to main test; temperature specification was slightly different (22°C, c.f. 24±1°C in main test).
- Any deformed or abnormal cells observed: None reported
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Test temperature:
24 ± 1°C
pH:
Nominal pH is approximately 8
Dissolved oxygen:
Not quantified - gaseous exchange was ensured by the action of the orbital shaker, oscillating at 120 cycles per minute.
Salinity:
Not applicable
Nominal and measured concentrations:
Nominal loadings: 0 (control), 0.46, 1.0, 2.2, 4.6, 10, 22, 46, and 100 mg/L.
Measured concentrations for diethylene glycol dibenzoate (expressed as % loading rate)
0.46 mg/L: 403 (Fresh prepared), 0 (expired / 96 hours old)
1.0 mg/L: 49.6, 0
2.2 mg/L: 79.3, 0
4.6 mg/L: 38.1, 0
10 mg/L: 36.7, 0
22 mg/L: 37.7, 0
46 mg/L: 32.2, 0
100 mg/L: 18.8, 0
An additional level at 10 mg/L DEGDB with no algae was prepared and found to contain 32.6% of loading in the fresh sample but with no detectable concentration after 96 hours.
Measured concentrations for diethylene glycol monobenzoate (expressed as % loading rate in terms of diethylene glycol dibenzoate)
0.46 mg/L: 40 (Fresh prepared), 0 (expired / 96 hours old)
1.0 mg/L: 5.3, 0
2.2 mg/L: 4.7, 0
4.6 mg/L: 4.3, 0
10 mg/L: 4.6, 0
22 mg/L: 4.8, 0
46 mg/L: 4.6, 15.8
100 mg/L: 5.0, 1.9
An additional sample at 10 mg/L DEGDB with no algae was prepared and found to contain 4.4% of loading in the fresh sample but with no detectable concentration after 96 hours.
A sampling error is suspected at the 0.46 mg/L level.
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): closed (loosely stoppered flasks)
- Material, size, headspace, fill volume: 250 mL flasks containing 100 mL medium.
- Aeration: Gaseous exchange and suspension of the algal cells were ensured by the action of the orbital shaker, oscillating at 120 cycles per minute.
- Renewal rate of test solution (frequency/flow rate): None (static test)
- Initial cells density: 1 x 10^4 cells/mL nominal, control cell density at 0h was 1.30 x 10^4 cells/mL.
- Control end cells density: 4.95 x 10^6 cells/mL
- No. of vessels per concentration (replicates):3
- No. of vessels per control (replicates):3

GROWTH MEDIUM
- Standard medium used: yes (Sterile nutrient medium as recommended in Official Journal No. L383A Part C3).

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Prepared in-house using deionised / reverse osmosis water.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: Not specified
- Photoperiod: Continuous illumination
- Light intensity and quality: 7000 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: electronic particle counter. Measurements taken at 0, 24, 48, 72, and 96 hours.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Approximately 2.2
- Range finding study
- Test concentrations: range finding studies conducted with initial loading rates of test substance ranging from 0.01 to 100 mg/L.
- Results used to determine the conditions for the definitive study: Yes
Reference substance (positive control):
no
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
2.7 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
cell number
Remarks on result:
other: 95% CL 2.32 - 3.04
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
3.3 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL 2.64 - 3.99
Duration:
96 h
Dose descriptor:
EL10
Effect conc.:
3.6 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
cell number
Remarks on result:
other: 95% CL 3.25 - 4.00
Duration:
96 h
Dose descriptor:
EL10
Effect conc.:
3.6 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL 2.66 - 4.86
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
5.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
cell number
Remarks on result:
other: 95% CL 4.92 - 5.58
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
10.94 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL 9.94 - 12.03
Duration:
96 h
Dose descriptor:
EL50
Effect conc.:
5.9 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
cell number
Remarks on result:
other: 95% CL 5.52 - 6.36
Duration:
96 h
Dose descriptor:
EL50
Effect conc.:
14.55 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL 12.67 - 16.70
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
cell number
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
2.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
96 h
Dose descriptor:
NOELR
Effect conc.:
2.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
cell number
Duration:
96 h
Dose descriptor:
NOELR
Effect conc.:
2.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): No abnormalities were detected in any of the cultures examined.
Bacterial contamination, observed in some of the test cultures at low levels, was not considered significant. The presence of bacteria during the study is thought to be due to the test substance being a biodegradable substrate for bacteria.
- Any stimulation of growth found in any treatment: A slight increase in area under the growth curve was seen in the 1 mg/L culture at 72 hours, and in the 1.0 and 4.6 mg/L cultures at 96 hours.
The analytical method used to confirm the concentrations of test material in solution determined concentrations of diethylene glycol monobenzoate (DEGDMB) as well as diethylene glycol dibenzoate (DEGDB); in general no detectable concentrations of either DEGDB or DEGMB were found in the expired samples (96 hours old).
Validity criteria fulfilled:
not specified
Conclusions:
The EL50 (Area under the curve 72 h) was 5.2 mg/L and the EL50 (Growth rate 0 - 72 h) was 11 mg/L, while the EL50 (Area under the curve 96 h) was 5.9 mg/L and the EL50 (Growth rate 0 - 96 h) was 15 mg/L.
Executive summary:

An algal growth inhibition test was conducted to determine the effect on the growth of algae of the test material diethylene glycol dibenzoate (DEGDB). The study was conducted according to EC, OECD, and US EPA test guidelines, and in compliance with GLP.

The 72h EL50 (Area under the curve, biomass) was 5.2 mg/L and the 72h EL50 (Growth rate) was 11 mg/L. The corresponding "No-observed effect loading rate" (NOELR) were 2.2 mg/L and 1.0 mg/L, respectively.

The 96h EL50 (Area under the curve, biomass) was 5.9 mg/L and the 96h EL50 (Growth rate) was 15 mg/L. The corresponding NOELR for both were 2.2 mg/L.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
7 November 1997 - 11 November 1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study undertaken at GLP accredited laboratory.
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OTS 797.1050 (Algal Toxicity, Tiers I and II)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Concentrations Nominal loadings: 0 (control), 0.10, 0.22, 0.46, 1.0, 2.2, 4.6 and 10 mg/L
- Sampling method: Duplicate samples (200 mL) were taken from control and test cultures at 0 hours and at 96 hours (replicates pooled) for analysis.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The method of preparation was selected following advice given in ECETOC 1996 Monograph No 26 as the test material has poor water solubility.
A stock solution of test material in chloroform (100 mg/mL) was prepared, then further stock solutions diluted using chloroform as required to give stock solutions at 46, 22, 10, 4.6, 2.2 and 1.0 mg/mL. 150 µL aliquots of each stock solution were added to autoclaved 2L vessels and the solvent was allowed to evaporate. 1.5 litres of algal medium were added to each vessel, then stirred for at least 18 hours using a magnetic stirrer with a 10 cm magnetic follower stirring at approximately 250 revolutions/minute. After stirring, the solutions were allowed to settle for approximately 1 hour, then a 500 mL (or 1 L for 1.0 mg/L level) aliquot of the water accomodated fraction was removed via a side tap from a mid water position.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): None reported
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Unicellular green algae
- Strain: No. CCAP 278/4
- Source (laboratory, culture collection): Culture Collection of Algae & Protozoa, Institute of Freshwater Ecology, Cumbria, UK
- Method of cultivation: Sterile nutrient medium was inoculated from a master culture and cultured under continuous illumination (c. 7000 lux) in an orbital incubator at 22°C to give an algal suspension in log phase growth, characterised by a cell density of 1.4E+07 cells / mL

ACCLIMATION
- Acclimation period:
- Culturing media and conditions (same as test or not): Lighting conditions used were equivalent to main test; temperature specification was slightly different (22°C, c.f. 24 ± 1°C in main test).
- Any deformed or abnormal cells observed: None reported
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Test temperature:
24 ± 1°C
pH:
7.3 to 7.4
Dissolved oxygen:
Not quantified - gaseous exchange was ensured by the action of the orbital shaker, oscillating at 120 cycles per minute.
Salinity:
Not applicable
Nominal and measured concentrations:
Nominal loadings: 0 (control), 0.10, 0.22, 0.46. 1.0, 2.2, 4.6, 10 mg/L
Measured concentrations for dipropylene glycol dibenzoate (expressed as % of loading rate)
0.10 mg/L : 99 (Fresh prepared), 50 (expired / 96 hours)
0.22 mg/L : 97, 52
0.46 mg/L: 82, 41
1.0 mg/L: 88, 53
2.2 mg/L: 88, 60
4.6 mg/L: 73, 66
10 mg/L: 69. 51
An additional level at 1.0 mg/L DPGDB with no algae was prepared and found to contain 86% of loading at 96 hours.
Measured concentrations for dipropylene glycol monobenzoate (expressed as % of loading rate dipropylene glycol dibenzoate)
0.10 mg/L : 6 (Fresh prepared), 24 (expired / 96 hours)
0.22 mg/L : 6, 23
0.46 mg/L: 5, 20
1.0 mg/L: 6, 20
2.2 mg/L: 6, 14
4.6 mg/L: 5, 11
10 mg/L: 6, 8
An additional level at 1.0 mg/L DPGDB with no algae was prepared and found to contain 6% of loading at 96 hours.
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): closed (loosely stoppered flasks)
- Material, size, headspace, fill volume: 250 mL flasks containing 100 mL medium.
- Aeration: Gaseous exchange and suspension of the algal cells were ensured by the action of the orbital shaker, oscillating at 120 cycles per minute.
- Renewal rate of test solution (frequency/flow rate): None (static test)
- Initial cells density: 1 x 10^4 cells/mL nominal, control cell density at 0h was 1.48 x 10^4 cells/mL.
- Control end cells density: 1.77 x 10^6 cells/mL
- No. of vessels per concentration (replicates):3
- No. of vessels per control (replicates):3

GROWTH MEDIUM
- Standard medium used: yes (Sterile nutrient medium as recommended in Official Journal No. L383A Part C3).

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Prepared in-house using deionised / reverse osmosis water.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: Not specified
- Photoperiod: Continuous illumination
- Light intensity and quality: 7000 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: electronic particle counter. Measurements taken at 0, 24, 48, 72, and 96 hours.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Approximately 2.2
- Range finding study
- Test concentrations: range finding studies conducted with initial loading rates of test substance ranging from 0.01 to 10 mg/L.
- Results used to determine the conditions for the definitive study: Yes
Reference substance (positive control):
no
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
0.15 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
cell number
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
0.89 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
96 h
Dose descriptor:
EL10
Effect conc.:
0.19 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
cell number
Duration:
96 h
Dose descriptor:
EL10
Effect conc.:
0.87 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
1.1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
cell number
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
4.9 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
96 h
Dose descriptor:
EL50
Effect conc.:
0.95 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
cell number
Duration:
96 h
Dose descriptor:
EL50
Effect conc.:
3.6 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
0.22 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
cell number
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
96 h
Dose descriptor:
NOELR
Effect conc.:
0.46 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): No abnormalities were detected in any of the cultures examined.
Bacterial contamination, observed in some of the test cultures at low levels, was not considered significant. The presence of bacteria during the study is thought to be due to the test substance being a biodegradable substrate for bacteria.
- Any stimulation of growth found in any treatment: A slight increase in growth rate was seen in the 0.10, 0.22 and 0.46 mg/L cultures at 72 hours, and in the 0.22 and 0.46 mg/L cultures at 96 hours.
The analytical method used to confirm the concentration of test material in solution determined concentrations of dipropylene glycol monobenzoate (DPGMB) as well as dipropylene glycol dibenzoate (DPGDB); the concentration of DPGDB was found to be lower after 96 hours than at the start of the test in all cases, whereas the concentration of DPGMB had increased in all samples. This observation suggests that the monobenzoate was a degradation product of the dibenzoate.
Validity criteria fulfilled:
yes
Conclusions:
The EL50 (Area under the curve 72 h) was 1.1 mg/L and the EL50 (Growth rate 0-72 h was 4.9 mg/L while the EL50 (Area under the curve 96 h) was 0.95 mg/L and the EL50 (Growth rate 0-96 h) was 3.6 mg/L.
Executive summary:

An algal growth inhibition test was conducted to determine the effect on the growth of algae of the test material dipropylene glycol dibenzoate (DPGDB). The study was conducted according to EC, OECD, and US EPA test guidelines, and in compliance with GLP.

The EL50 (Area under the curve 72 h) was 1.1 mg/L and the EL50 (Growth rate 0 - 72 h) was 4.9 mg/L. The corresponding "no-observed effect loading rates" (NOELR) were 0.22 and 1.0 mg/L, respectively. The EL50 (Area under the curve 96 h) was 0.95 mg/L and the EL50 (Growth rate 0 - 96 h) was 3.6 mg/L. A NOELR could not be obtained for Area under the curve 96 h, the NOELR for growth rate was 0.46 mg/L.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 November 1997 - 22 November 1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted in accordance with EC, OECD, and US EPA test guidelines, and in compliance with GLP.
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OTS 797.1050 (Algal Toxicity, Tiers I and II)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Concentrations Nominal loadings: 0 (control), 1.0, 2.2, 4.6, 10, 22, 46, and 100 mg/L
- Sampling method: Duplicate samples (100 mL) were taken from control and test cultures at 0 hours and at 96 hours (replicates pooled) for analysis. All media were filtered prior to analysis.
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The method of preparation was selected following advice given in ECETOC 1996 Monograph No 26 as the test material has poor water solubility.
A stock solution of test material in chloroform (1000 mg/mL) was prepared, then further stock solutions diluted using chloroform as required to give stock solutions at 460, 220, 100, 46, 22, 10 mg/mL. An aliquot, 200 µL, was added to each vessel and the solvent was allowed to evaporate. Two litres of algal medium were added to each vessel, then stirred for at least 18 hours using a magnetic stirrer with a 10 cm magnetic follower stirring at approximately 250 revolutions/minute. After stirring, the solutions were allowed to settle for approximately 1 hour, then a 500 mL aliquot (1 litre in the case of the 10 mg/L level) of the water accomodated fraction was removed via a side tap from a mid water position.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): None reported
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Unicellular green algae
- Strain: No. CCAP 278/4
- Source (laboratory, culture collection): Culture Collection of Algae & Protozoa, Institute of Freshwater Ecology, Cumbria, UK
- Method of cultivation: Sterile nutrient medium was inoculated from a master culture and cultured under continuous illumination (ca. 7000 lux) in an orbital incubator at 22 °C, to give an algal suspension in log phase growth, characterised by a cell density of 6.75x10E6 cells/mL.

ACCLIMATION
- Acclimation period:
- Culturing media and conditions (same as test or not): Lighting and temperature conditions used were equivalent to main test
- Any deformed or abnormal cells observed: None reported
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h
Test temperature:
24 °C
pH:
At 0 hours: 7.9 to 8.1
At 96 hours: 8.0 to 8.5
Dissolved oxygen:
Not quantified - gaseous exchange was ensured by the action of the orbital shaker, oscillating at 120 cycles per minute.
Salinity:
Not applicable
Nominal and measured concentrations:
Nominal loadings: 0 (control), 1.0, 2.2, 4.6, 10, 22, 46, and 100 mg/L.
Measured concentrations for triethylene glycol dibenzoate (expressed as % loading rate)
1.0 mg/L: 36 (Fresh prepared), 0 (expired / 96 hours old)
2.2 mg/L: 49, 0
4.6 mg/L: 47, 0
10 mg/L: 44, 0
22 mg/L: 32, 0
46 mg/L: 19, 0
100 mg/L: 13, 0
An additional level at 10 mg/L TEGDB with no algae was prepared and found to contain 45% of loading in the fresh sample but with no detectable concentration after 96 hours.
Measured concentrations for triethylene glycol monobenzoate (expressed as % loading rate in terms of triethylene glycol dibenzoate)
1.0 mg/L: 1 (Fresh prepared), 0 (expired / 96 hours old)
2.2 mg/L: 1, 2
4.6 mg/L: 1, 0
10 mg/L: 2, 0
22 mg/L: 1, 0
46 mg/L: 1, 0
100 mg/L: 1, 0
An additional sample at 10 mg/L TEGDB with no algae was prepared and found to contain 2% of loading in the fresh sample but with no detectable concentration after 96 hours.
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): closed (loosely stoppered flasks)
- Material, size, headspace, fill volume: 250mL flasks containing 100mL medium
- Aeration: Gaseous exchange and suspension of the algal cells were ensured by the action of the orbital shaker, oscillating at 120 cycles per minute.
- Initial cells density: 1 x 10^4 cells/mL nominal, control cell density at 0h was 1.11 x 10^4 cells/mL
- Control end cells density: Mean 5.32 x10^6
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3

GROWTH MEDIUM
- Standard medium used: yes (Sterile nutrient medium as recommended in Official Journal No. L383A Part C3)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Prepared in-house using deionised / reverse osmosis water.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: Not specified
- Photoperiod: continuous illumination
- Light intensity and quality: 7000 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: electronic particle counter; Measurements taken at 0, 24, 48, 72, and 96 hours.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Approximately 2.2
- Range finding study
- Test concentrations: range finding studies conducted with initial loading rates of test substance ranging from 0.01 to 100 mg/L.
- Results used to determine the conditions for the definitive study: Yes
Reference substance (positive control):
no
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
9.9 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
cell number
Duration:
72 h
Dose descriptor:
EL10
Effect conc.:
15 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
96 h
Dose descriptor:
EL10
Effect conc.:
9.3 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
cell number
Duration:
96 h
Dose descriptor:
EL10
Effect conc.:
24 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
33 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
cell number
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
96 h
Dose descriptor:
EL50
Effect conc.:
33 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
cell number
Duration:
96 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EL90
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
cell number
Duration:
72 h
Dose descriptor:
EL90
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
96 h
Dose descriptor:
EL90
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
cell number
Duration:
96 h
Dose descriptor:
EL90
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
cell number
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
22 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
96 h
Dose descriptor:
NOELR
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
cell number
Duration:
96 h
Dose descriptor:
NOELR
Effect conc.:
22 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): No abnormalities were detected in any of the cultures examined
Bacterial contamination, observed in some of the test cultures at low levels, was not considered significant. The presence of bacteria during the study is thought to be due to the test substance being a biodegradable substrate for bacteria.
- Any stimulation of growth found in any treatment: A slight increase in area under the growth curve was seen in the 1 mg/L and 10 mg/L cultures at 72 and 96 hours
The analytical method used to confirm the concentrations of test material in solution determined concentrations of triethylene glycol monobenzoate (TEGDMB) as well as triethylene glycol dibenzoate (TEGDB); in general no detectable concentrations of either TEGDB or TEGMB were found in the expired samples (96 hours old).
Validity criteria fulfilled:
yes
Conclusions:
The EL50 (Area under the curve 72h and 96h) for TEGDB was 33 mg/L and the EL50 (Growth rate 0 - 72h and 0 - 96h) was >100 mg/L.


Executive summary:

An algal growth inhibition test was conducted to determine the effect on the growth of algae of the test material triethylene glycol dibenzoate (TEGDB). The study was conducted according to EC, OECD and US EPA test guidelines, and in compliance with GLP

The EL50 (Area under the curve) after 72 and 96 hours for TEGDB was 33 mg/L and the EL50 (Growth rate) from 0-72 hours and 0-96 hours was >100 mg/L. The "No-observed effect loading rate" (NOELR) was 10 mg/L.

Description of key information

DEGDB:

Toxicity to aquatic algae and cyanobacteria:

- Key study, reliability 1, OECD 201, Pseudokirchneriella subcapitata - The 72h EL50 (Area under the curve, biomass) was 5.2 mg/L and the 72h EL50 (Growth rate) was 11 mg/L. The corresponding "No-observed effect loading rate" (NOELR) were 2.2 mg/L and 1.0 mg/L, respectively. The 96h EL50 (Area under the curve, biomass) was 5.9 mg/L and the 96h EL50 (Growth rate) was 15 mg/L. The corresponding NOELR for both were 2.2 mg/L.

DPGDB:

Toxicity to aquatic algae and cyanobacteria:

- Key study, reliability 1, OECD 201, Pseudokirchneriella subcapitata - The EL50 (Area under the curve 72 h) was 1.1 mg/L and the EL50 (Growth rate 0 - 72 h) was 4.9 mg/L. The corresponding "no-observed effect loading rates" (NOELR) were 0.22 and 1.0 mg/L, respectively. The EL50 (Area under the curve 96 h) was 0.95 mg/L and the EL50 (Growth rate 0 - 96 h) was 3.6 mg/L. A NOELR could not be obtained for Area under the curve 96 h, the NOELR for growth rate was 0.46 mg/L.

TEGDB

Toxicity to aquatic algae and cyanobacteria:

- Key study, reliability 1, OECD 201, Pseudokirchneriella subcapitata - The EL50 (Area under the curve 72h and 96h) for TEGDB was 33 mg/L and the EL50 (Growth rate 0 - 72h and 0 - 96h) was >100 mg/L. The "No-observed effect loading rate" (NOELR) was 10 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:
4.9 mg/L
EC10 or NOEC for freshwater algae:
1 mg/L

Additional information

This substance is a reaction mass of dipropylene glycol dibenzoate (DPGDB), diethylene glycol dibenzoate (DEGDB) and triethylene glycol dibenzoate (TEGDB). No testing has been performed on the reaction mass itself but data are available for DPGDB, DEGDB and TEGDB individually. All the studies were performed according to international test guidelines and in compliance with GLP. They are presented below, by component. 

The algal growth inhibition studies were performed on Selenastrum capricornutum (Pseudokirchnerella subcapitata). The substance was introduced using a water accommodated fraction (WAF). In general the WAF studies suffered from low concentrations being measured in the initial WAF solution and subsequent losses of the test substance.

These data demonstrate that DPDGB and DEGDB have similar environmental profiles with all EC/EL/LC/LL50 values in the range of >1.0 mg/L and <20 mg/L. TEGDB is less toxic with EC/EL/LL50 values in excess of 16 mg/L.

The most sensitive endpoint result obtained for these components has been used as the key value to evaluate hazard and is highlighted below. Where no data exist an overall statement that applies to the reaction mass has been made. No further testing is proposed.

The key value is:

Toxicity to aquatic algae and cynaobacteria EC50 = 4.9 mg/L

 

These values are from DEGDB, acute toxicity to algae performed on any of the components.

 

DEGDB

Toxicity to aquatic algae and cyanobacteria

The EL50 (Area under the curve 72h) was 5.2 mg/L and the EL50 (Growth rate 0 – 72 h) was 11 mg/L while the EL50 (Area under the curve 96 h) was 5.9 mg/L and the EL50 (Growth rate 0 - 96 h) was 15 mg/L. (HLS 2001, VCL265/974223).

DPGDB

Toxicity to aquatic algae and cyanobacteria

The EL50 (Area under the curve 72 h) was 1.1 mg/L and the EL50 (Growth rate 0 - 72 h) was 4.9 mg/L while the EL50 (Area under the curve 96 h) was 0.95 mg/L and the EL50 (Growth rate 0 - 96 h) was 3.6 mg/L. (HLS 2001, VCL 253/974220).

TEGDB

Toxicity to aquatic algae and cyanobacteria

The EL50 (Area under the curve 72h and 96h) was 33 mg/L and the EL50 (Growth rate 0 – 72 h and 0 – 96 h) was >100 mg/L (HLS 2001, VCL284/974409).