Registration Dossier

Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

Administrative data

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.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report Date:
2001

Materials and methods

Test guidelineopen allclose all
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Benzoflex 9-88 (Dipropylene glycol dibenzoate DPGDB)
- Physical state: Clear colourless liquid
- Analytical purity: Dipropylene glycol dibenzoate - 89.4% (w/w)
- Impurities (identity and concentrations):
Dipropylene glycol monobenzoate - 4.98% (w/w)
Propylene glycol dibenzoate - 2.29% (w/w)
Propylene glycol monobenzoate - 0.28% (w/w)
Propenyloxy propyl benzoate - 2.35% (w/w)
- Lot/batch No.: 56635211
- Expiration date of the lot/batch: 17 December 1997
- Storage condition of test material: Room temperature

Sampling and analysis

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.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The method ofpreparation 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

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

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h

Test conditions

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

Results and discussion

Effect concentrationsopen allclose all
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.

Applicant's summary and conclusion

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.