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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2003-11-18 to 2003-11-21
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The study was conducted according to the appropriate OECD guideline, in accordance with GLP and analytical monitoring was carried out. However in all the test concentrations bright green dispersions were observed by the end of the test period and in the highest 3 concentrations clumps of algal cells were noted. At the nominal concentrations of 32 mg/l and below no algal clumps were recorded. Therefore a more reliable result is based on these concentrations, leading to an EC50 value at concentrations at which no clumps were observed
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Water samples were taken from the control (replicates RI - R3 pooled) and each test group (replicates RI - R3 pooled) at 0 and 72 hours for quantitative analysis.

- Sampling method: direct from vessels

- Sample storage conditions before analysis: Duplicate samples were taken at 0 hours and stored (approximately -20°C) for further analysis if necessary. Sample volumes required for chemical analysis precluded the storage of duplicate samples at 72 hours.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)

- Method: Amounts of test material (640 and 200 mg) were each separately dissolved in culture medium and the volumes adjusted to 2 litres to give 320 and 100 mg/lL stock solutions respectively. A series of dilutions was made from these stock solutions to give further stock solutions of 32, 10, 3.2, 1.0 and 0.32 mg/L. An aliquot (1 litre) of each of the stock solutions was separately inoculated with algal suspension (5.0 ml) to give the required test concentrations of 0.32, 1.0,3.2, 10,32, 100 and 320 mg/L.

- Controls: culture medium
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM

- Strain: CCAP 276/20

- Source (laboratory, culture collection): from the Culture Collection of Algae and Protozoa (CCAP), Institute of Freshwater Ecology, The Ferry House, Far Sawrey, Ambleside, Cumbria. Cultures were maintained in the laboratory by the periodic replenishment of culture medium.

- Age of inoculum (at test initiation):
- Method of cultivation:

ACCLIMATION
- Acclimation period:
- Culturing media and conditions (same as test or not):
- Any deformed or abnormal cells observed:
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
24 ± 1°C
pH:
range: 7.3 to 8.1 in the controls
Nominal and measured concentrations:
Nominal concentrations: 0.32, 1.0, 3.2, 10, 32, 100 and 320 mg/l
Details on test conditions:
TEST SYSTEM

- Test vessel: flasks

- Material, size, headspace, fill volume: 250 ml conical flasks, filled with 100 ml test medium

- Initial cells density: 1 x 10^4 cells/ml

- Control end cells density: 5.89 x 10^5 cells per ml

- No. of vessels per concentration (replicates): 3

- No. of vessels per control (replicates): 3


GROWTH MEDIUM

- Standard medium used: yes


TEST MEDIUM / WATER PARAMETERS

- Source/preparation of dilution water: dechlorinated tap water

- Culture medium different from test medium: no

- Intervals of water quality measurement: pH was measured at 0 h and at 72 h. The temperature of the incubator was recorded daily.


OTHER TEST CONDITIONS

- Sterile test conditions: no

- Adjustment of pH: none reported

- Photoperiod: continuous illumination

- Light intensity and quality: approx 7000 lux


EFFECT PARAMETERS MEASURED:

- Determination of cell concentrations: electronic particle counter, every 24 h


TEST CONCENTRATIONS

- Spacing factor for test concentrations: 3

- Range finding study: two range finding studies were conducted, the first results the results obtained showed a flat response in terms of inhibition of growth rate. Both tests tested the same concentrations.

- Test concentrations: 0.10, 1.0, 10 and 100 mg/L

- Results used to determine the conditions for the definitive study: no statistically significant decrease in gell cell growth was observed at 0.1 and 1.0 mg/L, however growth was observed to be reduced at 10 and 100 mg/L.
Reference substance (positive control):
no
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 17 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
1.8 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 32 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
3.2 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): see Any other information on material and methods section

- Flocculation: see Any other information on material and methods section

- Aggregation of algal cells: After 72 hours there were no abnormalities detected in the control or test cultures at 0.32, 1.0, 3.2 and 10 mg/l, however cell clumping was observed in the test cultures at 32, 100 and 320 mg/L.


- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: see Any other information on material and methods section

- Effect concentrations exceeding solubility of substance in test medium: the test substance hydrolyses rapidly.
Reported statistics and error estimates:
Dunnett's multiple comparison procedure (1995), using SAS computer software package.
It was not possible to calculate 95% confidence limits for the ECM values as the data generated did not fit the models available for the calculation of confidence limits.

Analytical monitoring:

Analysis of the test solutions at 0 hours showed measured test concentrations of the parent test material to ranged from 32% to 58% of nominal. The variable results obtained were considered to be due to the rapid hydrolysis of the parent test material. Analysis of the test solutions at 72 hours showed a marked decline in measured test concentrations of parent test material with concentrations ranging from less than the limit of quantitiation (LOQ) of the analytical method employed to 0.1% of nominal. This decline was due to the instability of the test material in culture medium and was in-line with the preliminary stability analyses conducted which indicated that the test material was unstable.

The hydrolysis product of the substance is subject to further hydrolysis (epoxide ring opening). It is the opinion of the reviewer that this explains the difference between measured and expected hydrolysis product concentration.

Table 1. Analytical results

      Hydrolysis product  Parent test material  Total Peak Area  Geometric mean hydrolysis product  Geometric mean parent substance
Sample  Nominal concentration (mg/l)   Concentration found (mg/l) Expressed as % of nominal  Concentration found (mg/l)  Expressed as % nominal   Concentration found (mg/l) Expressed as % nominal  0 -72 h (mg/l)   0 -72 h (mg/l)
 0 hours  Control  <LOQ  -  <LOQ  -  <LOQ  -  -  -
 0.32  <LOQ  -  0.104  32  0.104  32    
 1.0  0.006  0.6  0.599 56   0.565  57    
 3.2  0.103  3  1.82  57  1.92  60    
 10  0.541  5  5.67  57  6.21  62    
 32  2.30  7  17.1  54  19.4  61    
 100  6.61  7  57.9  58  64.5  64    
 320  24.8  8  165  52  190  59    
 72 hours  Control  <LOQ  -  <LOQ  -  <LOQ  -  -  -
 0.32  <LOQ  -  <LOQ  -  <LOQ  -  -  -
 1.0  <LOQ  -  <LOQ  -  <LOQ  -  -  -
 3.2  -  -  <LOQ  -  <LOQ  -  -  -
 10  <LOQ  -  <LOQ  -  <LOQ  -  -  -
 32  <LOQ  -  <LOQ  -  <LOQ  -  -  -
 100  <LOQ  -  0.125  0.1  0.125  0.1  -  2.7
 320  <LOQ  -  0.277  0.1  0.277  0.1  -  6.8

Table 2. Cell densities and percentage growth inhibition.

 Nominal concentration (mg/L)  Area under the curve at 72 h  % inhibition  Growth rate (0 -72 h)  % inhibition
 Control  1.63 x10^7  -  0.057  -
 0.32  1.66 x10^7  [2]  0.058  [2]
 1.0  1.66 x10^7  [2]  0.058  [2]
 3.2  1.25 x10^7  24  0.053  7
 10  8.22 x10^6  50  0.043  25
 32  8.78 x 10^6  46  0.046  19
 100 7.04 x10^6  57  0.038  33
 320  3.07 x10^6  81  0.026  54
Validity criteria fulfilled:
yes
Conclusions:
A 72 h ErC50 nominal value of 280 mg/L, equivalent to 2.7 to 6.8 mg/l parent substance (geom. mean), and a NOEC nominal value of 1.0 mg/L (geometric mean not available) have been determined for the effects of the test substance on the growth rate of the freshwater algae Scenedesmus subspicatus. In all the test concentrations bright green dispersions were observed by the end of the test period and in the highest 3 concentrations clumps of algal cells were noted. It cannot be excluded that the observed effects are related to undispersed test material, attributable by the reviewer to the formation of less soluble tetramers of the hydrolysis product of the registered substance. Therefore the results of the study should be treated with caution. At the nominal concentrations of 32 mg/l and below no algal clumps were recorded. Therefore a more reliable EC50 should be based on these concentrations. A 72 h ErC50 value of >32 mg/l nominal, equivalent to >17 mg/l measured initial (geometric mean not possible), and a NOEC of 3.2 mg/l nominal, 1.8 mg/l, measured initial, have thus been estimated by the current reviewer; where 19% and 7% inhibition were recorded at 17 and 1.8 mg/l respectively.

Description of key information

Short-term toxicity to algae: (72 h) ErC50: >32 mg/l and NOEC 3.2 mg/l (nominal) (OECD 201). The EC50 is equivalent to >25 mg/l when expressed in terms of the hydrolysis product [3-(2,3-epoxypropoxy)propyl]methylsilanediol.

Key value for chemical safety assessment

EC50 for freshwater algae:
25 mg/L
EC10 or NOEC for freshwater algae:
2.5 mg/L

Additional information

A 72 h ErC50 nominal value of 280 mg/l, equivalent to 2.7 to 6.8 mg/l parent substance (geometric mean), and a NOEC nominal value of 1.0 mg/l (geometric mean not available) have been determined for the effects of the test substance on the growth rate of the freshwater algae Scenedesmus subspicatus. In all the test concentrations bright green dispersions were observed by the end of the test period and in the highest 3 concentrations clumps of algal cells were noted. It cannot be excluded that the observed effects are related to undispersed test material, attributable by the reviewer to the formation of less soluble tetramers of the hydrolysis product of the registered substance. Therefore the results of the study should be treated with caution, however the results are taken forward for use in the chemical safety assessment as they represent a conservative worst case. At the nominal concentrations of 32 mg/l and below no algal clumps were recorded. Therefore a more reliable EC50 should be based on these concentrations. A 72 h ErC50 value of >32 mg/l nominal, equivalent to >17 mg/l measured initial (geometric mean not possible), and a NOEC of 3.2 mg/l nominal, 1.8 mg/l, measured initial, have thus been estimated by the current reviewer; where 19% and 7% inhibition were recorded at 17 and 1.8 mg/l respectively.

Epoxides are known to have the potential for enhanced toxicity due to a specific mode of action to higher organisms (e.g. by Lipnick, 1991); therefore even though the available experimental data provide an assessment limited to a relatively low concentration, the assessment is driven by the data on invertebrates and fish and the uncertainties in interpretation of this test result are not considered critical to the data set.

 

The study report discusses the stability of the test substance in water and reports that the half-life of the test material was determined to be approximately 2 hours. The stability analysis also showed that the hydrolysis products themselves were possibly unstable. Therefore it was determined that the test organisms would be exposed to the parent test material and hydrolysis products, therefore the effects seen in the test are attributed to [3-(2,3-epoxypropoxy)propyl]methylsilanediol. In view that the analytical results are for parent substance, it is appropriate to interpret the results in terms of nominal concentration.

 

The results may be expressed in terms of concentration of the hydrolysis product, (2,3-epoxypropoxy)propyl]methylsilanediol, by applying a molecular weight correction: (MW of silanol = 192.28 / MW of parent = 248.40) * >32 mg/l = >25 mg/l. The NOEC value is converted using the same method.

 

[3-(2,3-epoxypropoxy)propyl]methylsilanediol is susceptible to further hydrolysis reactions and the ultimate hydrolysis product 3-{3-[dihydroxy(methyl)silyl]propoxy}propane-1,2-diol is considered unlikely to exhibit significant ecotoxic effects based on QSAR estimated E(L)C50s >>100 mg/l (ECOSAR). The rate of reaction under environmental conditions is uncertain but the half-life is not expected to be more than a few days.

 

Reference

Lipnick RL. 1991. Outliers: their origin and use in the classification of molecular mechanisms of toxicity. Sci. Total. Environ. 109-110:131153.