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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:
from 2000-02-15 to 2000-02-18
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study. The study was conducted according to the equivalent OECD guideline. It was conducted according to GLP however no analytical monitoring was carried out.
Qualifier:
according to guideline
Guideline:
ISO 10253 (Water quality - Marine Algal Growth Inhibition Test with Skeletonema costatum and Phaeodactylum tricornutum)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION

- Method: Single WAFs were prepared by adding 0, 30.6, 96.6, 302.4, 929.1 and 3030.3 mg of test substance to seawater and stirring for 20 h in the dark. After a phase separation period of approx. 4 hours, the clear middle layer of each extract was siphoned off.

- Controls: seawater only, negative control. A background series containing test substance without algae was also used to measure background particle density.

- Evidence of undissolved material (e.g. precipitate, surface film, etc): the test media appeared clear throughout the study and at all treatment levels
Test organisms (species):
Skeletonema costatum
Details on test organisms:
TEST ORGANISM

- Source (laboratory, culture collection): IRCHA, BP 1, F-91350 Vert-le-Petit, France

- Age of inoculum (at test initiation): 3 to 4 days old

- Method of cultivation: not reported


ACCLIMATION

- Acclimation period: not reported

- Culturing media and conditions (same as test or not): same culturing medium

- Any deformed or abnormal cells observed: not reported
Test type:
static
Water media type:
saltwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
not reported
Test temperature:
20+/-2°C
pH:
range:8.0-9.0

The increase of pH is caused by the algae growth in the test vessels (this is confirmed by measurement of pH in the test background particle test system with no cells). In common with other species of marine algae, Skeletonema costatum gains CO2 from HCO3-, leading to a release of hydroxide ions, that raise the alkalinity of the medium.

The shift in pH of >1 unit is indicative of good algal growth and is not considered to invalidate the test result.
Dissolved oxygen:
n/a
Salinity:
30‰
Nominal and measured concentrations:
Nominal loading rate: 0, 10, 31.5, 98.6, 316 and 988 mg/l
Details on test conditions:
TEST SYSTEM

- Test vessel: conical flasks

- Type (delete if not applicable): closed, with silicone sponge caps

- Material, size, headspace, fill volume:
- Aeration: no

- Initial cells density: 0.35 x 10^3 particles/ml

- Control end cells density:

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

- No. of vessels per control (replicates): 6


GROWTH MEDIUM

- Standard medium used: yes


TEST MEDIUM / WATER PARAMETERS

- Source/preparation of dilution water: natural sea water with a salinity of 30‰ sterilized by micropore filtration. Nutrients were added to the medium via concentrated stock solutions.

- Culture medium different from test medium: no

- Intervals of water quality measurement: pH was measured at the start and after 70.5 hours

OTHER TEST CONDITIONS

- Sterile test conditions: yes

- Adjustment of pH: no

- Photoperiod: Continuous

- Light intensity and quality: 60-120 umol/s/m

- Salinity (for marine algae): 30‰


EFFECT PARAMETERS MEASURED

- Determination of cell concentrations: electronic particle counter, Coulter Multisizer model IIe, measured at 0, 25, 48 and 70.5 h. The morphology of the algae was examined visually with the aid of a microscope at the start and end of the test.


TEST CONCENTRATIONS

- Spacing factor for test concentrations: 3
Reference substance (positive control):
yes
Remarks:
potasssium dichromate and/or 3.5-dichlorophenol
Duration:
70.5 h
Dose descriptor:
NOELR
Effect conc.:
>= 988 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
WAFs
Basis for effect:
growth rate
Duration:
70.5 h
Dose descriptor:
EL50
Effect conc.:
> 988 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
(WAFs)
Basis for effect:
growth rate
Results with reference substance (positive control):
- Results with reference substance valid? yes

- EC50: not reported

- Other: the test system used is checked with the references substances on a regular basis. The results of these reference tests are in agreement with the mean EC50 values found in international ring tests.
Reported statistics and error estimates:
The data were analysed using the methods of Kooijman et al.

None of the growth parameters were inhibited by more than 50% over the course of the test. It was therefore not possible to determine EL50 values.

Growth was not adversely affected in any of the treatments. The NOEL was therefore equivalent to the highest treatment level.

Table 1. Mean particle counts (x103particles/ml) corrected for background

 

 

Loading rate (mg/l)

Time (h)

0

0

10

31.5

98.6

316

988

0

0.4

0.3

0.4

0.4

0.4

0.4

0.4

25

1.9

1.7

1.6

1.6

1.8

1.8

1.8

48

34.9

30.1

33.4

33.4

35.0

36.6

37.4

70.5

166.9

166.0

131.3

122.2

138.7

132.4

129.9

 

Table 2. Individual growth parameters calculated from the results of the test

 

Loading rate (mg/l)

Calculated inoculum (x106/μm3/ml)

Calculated growth rate (h-1)

Calculated yield (x106/μm3/ml)

0

0.03

0.1384

130

10

0.03

0.1441

107

31.5

0.02

0.1555

101

98.6

0.02

0.1526

110

316

0.02

0.1564

108

988

0.02

0.1524

108

 

Table 3. Area under the growth curve (A) and % inhibition of growth (IA)

 

 

Loading rate (mg/l)

Parameter

0

0

10

31.5

98.6

316

988

A

2694

2573

2263

2151

2377

2345

2334

IA

0

0

14

18

10

11

11

 

Validity criteria fulfilled:
yes
Conclusions:
A 70.5-hour EL50 of >988 mg/l and NOELR of ≥988 mg/l have been determined for the effects of the test substance on growth (expressed as area under the growth curve (A)) of the marine algal species Skeletonema costatum. These values are above the limit of water solubility (LoS) of the substance.
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2000-10-16 to 2000-10-20
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Qualifier:
according to guideline
Guideline:
EPA OTS 797.1050 (Algal Toxicity, Tiers I and II)
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Concentrations: One replicate was reserved for analytical sampling at 0-, 24- and 96-hour.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION

- Method: A 0.20 mg a.i./mL stock solution was prepared by placing 0.0201 g (0.0200 g as active ingredient) of D5 in a 100?mL volumetric flask and diluting to volume with acetone (CAS No. 67-64-1). To minimize the potential for volatilization of the test substance, each 20 µg a.i./L nominal test solution was prepared by directly adding 0.027 mL of the 0.20 mg a.i./mL primary stock solution to the sterile AAP medium (containing an additional 500 mg/L of sodium bicarbonate) in each 265-mL replicate flask.

Growth/test medium: The culture medium used was Algal Assay Procedure (AAP) medium prepared with sterile, deionized water. AAP medium used to prepare the exposure solutions was formulated in the same manner as the culture medium except that an additional 500 mg/L of sodium bicarbonate was added to the medium to provide sufficient dissolved bicarbonate for cell growth in a closed system.

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM

- Strain: Pseudokirchneriella subcapitata, formerly Selenastrum capricornutum, strain 1648, Class Chlorophyceae.

- Source (laboratory, culture collection): The alga was obtained from the University of Texas, Austin, Texas, and was maintained in stock culture at Springborn Smithers.


ACCLIMATION

- Culturing conditions: The stock cultures were maintained within the following conditions: a shaking rate of 100 ± 10 rpm, a temperature of 24 ± 1 °C and continuous illumination at the surface of the medium with an intensity of approximately 300 to 500 foot candles (3200 to 5400 lux). Lighting was supplied by Duro-Test® Vita-Lite® fluorescent bulbs. Culture flasks were agitated continuously on an orbital shaker.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h
Hardness:
no data
Test temperature:
24 °C
pH:
pH measured in the dilution water and solvent control vessels was 7.5 at test initiation and 9.4 at test termination.

pH measured in the 20 µg a.i./L treatment level was 7.5 at test initiation and 9.4 at test termination.
Dissolved oxygen:
no data
Salinity:
not applicable
Nominal and measured concentrations:
Nominal concentrations in µg/L: 20

Initial measured concentrations in µg/L: 12

Unit [results expressed in what unit]: µg a.i./L

The results are reported and interpreted with reference to initial measured concentration
Details on test conditions:
TEST SYSTEM

- Test vessel: The test was conducted in sterile 265-mL Erlenmeyer flasks containing 265 mL of test solution. All test vessels were fitted with a Teflon®-lined screw cap.

- Test Design: Each flask was filled to approximately 90% of the final volume; a 1.3-mL inoculum of Pseudokirchneriella subcapitata cells, at a density of approximately 211 x 10E4 cells/mL, was aseptically introduced into each flask. This inoculum provided the required initial (0 hour) cell density of approximately 1.0 x 10E4 cells/mL. Each flask was then filled entirely (approximately 265 mL) with the appropriate test or control solution, yielding zero headspace. Each flask was immediately capped with a Teflon®-lined screw cap. Seventeen replicate test vessels were established for the treatment level, the dilution water control and the solvent control (three replicates for cell counts at each interval; 24-, 48-, 72- and 96-hour; one replicate for analytical sampling at 0-, 24- and 96-hour; one replicate for water quality at 0- and 96-hour).


- Water chemistry in test: TOC concentration of the AAP sample collected in October 2000 was 0.59 mg/L. The dilution water and solvent control vessels both had a specific conductivity range of 80 to 90 mmhos/cm at test initiation and at test termination. The 20 µg a.i./L treatment level had a specific conductivity of 80 µmhos/cm at test initiation and 90 µmhos at test termination.

- Light levels and quality during exposure: 380 - 500 foot candles (4100 - 5400 lux). The photosynthetically-active radiation (PAR) of the test area measured at test initiation ranged from 56 to 73 µE/m2/s.
Reference substance (positive control):
not specified
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
>= 12 µg/L
Nominal / measured:
meas. (initial)
Conc. based on:
act. ingr.
Basis for effect:
other: cell density and growth rate
Duration:
96 h
Dose descriptor:
EC10
Effect conc.:
> 12 µg/L
Nominal / measured:
meas. (initial)
Conc. based on:
act. ingr.
Basis for effect:
other: cell density and growth rate
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
> 12 µg/L
Nominal / measured:
meas. (initial)
Conc. based on:
act. ingr.
Basis for effect:
other: cell density and growth rate
Remarks on result:
other: not calculable
Details on results:
- Exponential growth in the control (for algal test): yes
Reported statistics and error estimates:
Statistical methods: A t-Test was used to compare the cell density and growth rate of the control to that of the solvent control. A t-Test was also used to compare the cell density and growth rate of the treatment level to the pooled control.

Table 1 Results of analysis of test media

 Nominal test substance concentration (μg/L)  Measured concentration at start of test (μg/L) Percent of nominal   Measured concentration after 24 hours (μg/L)  Percent of nominal   Measured concentration after 96 hours (μg/L)  Percent of nominal
 0 (Control)  <0.50  -  <0.10  -  <0.50  -
 0 (Solvent control)  <0.50  -  <0.10  -  <0.50  -
 20  12*  62  9.2*  47  2.1*  11

*Mean of three replicates

Table 2. Test results

 Initial mean measured concentration (μg/L)  Mean initial cell density (cells/mL)  Mean cell density after 24 hours (cells/mL)   Mean cell density after 48 hours (cells/mL)  Mean cell density after 72 hours (cells/mL)    Mean cell density after 96 hours (cells/mL) Percent inhibition  Growth rate 0 -72 h (/day)  Percent inhibition 
 0 (Control)  10000  89000  620000  1090000  2700000  -  1.61 (0.009)
 0 (Solvent control)  10000  100000  580000  1120000  2690000  1.62 (0.008)  -
Pooled control  -  -  -  -  2700000  1.61 (0.009)  -
 12  10000  69000  530000  1110000  2690000  0.19 1.61 (0.009)   0.00

Cell densities are means of three replicates

Standard deviations are shown in brackets

Validity criteria fulfilled:
yes
Conclusions:
A 96-hour ErC50 of >12 µg a.i./L has been determined for the effects of the test substance on growth rate of Selenastrum capricornutum. A NOEC of ≥12 µg a.i./L has been determined in the same test.

Additional testing was not conducted to further define the EC values since the nominal concentration tested was
slightly above the water solubility limit of the test substance.
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2006-12-11 to 2006-12-15
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Although test substance concentrations in the solvent stock solutions were measured prior to the start of the study, concentrations in the test vessels were not measured. Methods to maintain test concentrations (e.g. flow-through) in algae studies are unavailable, hence this study is representative of the potential effect on algae initially exposed to the test substance but subsequently and progressively exposed to its hydrolysis products.
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
At test initiation, a single sample was removed from each treatment level stock solution prepared (i.e. 1.0, 2.0, 4.0, 8.0 and 16 mg/mL. The samples were analysed for the test substance.
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION

- Method: A 16 mg/mL stock solution was prepared prior to test initiation by placing 0.4001 g of the test substance in a 25-mL volumetric flask and bringing it to volume with acetone. Test solutions were prepared from dilutions of the 16 mg/mL stock solution using AAP medium.

- Controls: Algal growth medium and algal growth medium plus acetone (0.1 mL/L)
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM

Test organism: Pseudokirchneriella subcapitata

Source: Springborn Smithers culture

Age of inoculum: 3 days since previous transfer
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
Not reported
Test temperature:
24ºC
pH:
pH at test initiation: 7.0 to 7.2

pH at test termination: 9.2 to 9.3 (the increase in pH during exposure is common in static algal cultures and is due to photosynthesis by the algae)
Dissolved oxygen:
Not reported
Salinity:
Not applicable
Nominal and measured concentrations:
Nominal test concentrations: 0 (Control), 0 (Solvent control), 0.10, 0.20, 0.40, 0.80 and 1.6 mg/L.  The concentration of 1.6 mg/L, the highest concentration tested, is considered representative of the functional limit of solubility.  All test solutions were observed to be clear and colorless with no visible undissolved test substance.
Details on test conditions:
TEST SYSTEM

- Test conditions: Closed system. Due to the volatile nature of the test substance volatile organic analysis (VOA) vials with Teflon ®-lined screw caps were used rather than traditional caps which allow air exchange.  This procedure was used to minimize the loss of test substance.  

- Twelve replicates per treatment level and the control and 24 replicates for the solvent control were prepared.  The additional two replicates per treatment level and controls were used for analytical and water quality measurements.  

- Initial cell density: 1.5x10E4 cells/mL.

- Water quality parameters (pH and conductivity) were measured at test initiation and at the termination of the 96-hour exposure period.

- Dilution water: Algal Assay Procedure (AAP) medium

- Continuous illumination at 420 to 540 footcandles (4500 to 5800 lux), shaking rate of 100 rpm.  

- Effect criteria: 0- to 96-hour cell density and 0- to 72-hour average growth rate (μ) and biomass expressed as yield relative to the performance of the solvent control data.

- 0 to 96-hour cell density and 0 to 72-hour average growth rate (μ ave) and biomass expressed as yield were determined relative to the performance of the solvent control. Yield was calculated as biomass (cell density) at each interval of the test minus the initial biomass at the start of the test. 
Reference substance (positive control):
no
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 1.6 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: It is likely that the test organisms were predominantly exposed to the hydrolysis products of the test substance.
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
>= 1.6 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: It is likely that the test organisms were predominantly exposed to the hydrolysis products of the test substance.
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 1.6 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: It is likely that the test organisms were predominantly exposed to the hydrolysis products of the test substance.
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 1.6 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Biomass expressed as yield
Remarks on result:
other: It is likely that the test organisms were predominantly exposed to the hydrolysis products of the test substance.
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 1.6 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Biomass expressed as yield
Remarks on result:
other: It is likely that the test organisms were predominantly exposed to the hydrolysis products of the test substance.
Details on results:
- Exponential growth in the control (for algal test): yes
Reported statistics and error estimates:
No treatment resulted in >50% inhibition of growth rate or biomass. It was therefore not possible to determine definitive EC50 values for these end points.

No observed effect concentrations (NOECs) for growth rate and biomass (yield) were determined using Bonferroni's t-Test. The calculations were performed using TOXSTAT® version 3.5 software.

Table 1. Test results

 Nominal concentration (mg/L)  Initial cell concentration (cells/mL)  Cell concentration after 24 hours (cells/mL)   Cell concentration after 48 hours (cells/mL)  Cell concentration after 72 hours (cells/mL)  Inhibion of growth rate after 72 hours (%)   Inhibion of biomass (yield) after 72 hours (%)  
 0 (Control)  15000  51700  194200  399200  -  -
 0 (Solvent control)  15000  56700  172900  406700  -  -
 0.10  15000  62500  181700  368300  3  10
 0.20  15000  51700  177500  348300  5  15
 0.40  15000  55000  192500  394200  1  3
 0.80  15000  54200  192500  323300  7  21
 1.6  15000  54200  176700  375800  3  8


The following acceptance criteria are required by OECD Guideline 201: the cell growth in the solvent control must increase
from the initial density (1.5 x 10E4 cells/mL) by more than 16 times after 72 hours of growth.  Additionally, the mean
coefficient of variation (CV) for section-by-section specific growth rates (day 0 to 1, 1 to 2, and 2 to 3) in the solvent control replicates should not exceed 35%.  The CV for the average growth rate of the solvent control for
the entire test period (0- to 72-hour growth rate) should not exceed 7%.  During this study, the 72-hour cell density
in the solvent control was 39.92 x 10E4 cells/mL, which meets the criterion of 24 x 10E4 cells/mL at 72 hours.  The
mean daily CV for growth rates observed in the test was 28% and the CV for 0- to 72-hour growth rate was 2.6%. These values are within the acceptability criteria.

Validity criteria fulfilled:
yes
Conclusions:
A 72-hour EC50 value of >1.6 mg/L and NOEC of ≥1.6 mg/L have been determined for the effects of the test substance on growth rate of Pseudokirchnerella subcapitata. A concentration of 1.6 mg/L corresponds to the approximate water solubility of the substance. It is likely that the test organisms were predominantly exposed to the hydrolysis products of the substance.
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
14 Feb - 17 Feb 2006
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study is considered to be reliability 2 (reliable with restrictions) because some observations associated with validity criteria in the equivalent OECD test guideline are not reported. Read-across of the study is considered to be reliability 2.
Qualifier:
according to guideline
Guideline:
other: Circular on Test Methods of New Chemical Substances (Japan), Alga growth inhibition test
Deviations:
no
GLP compliance:
yes
Remarks:
according to the Japanese GLP Standard
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material
PHYSICO-CHEMICAL PROPERTIES
- Melting point: < 0°C
- Boiling point: 123°C
- Vapour pressure: < 2.7 kPa at 25 °C
- Water solubility (under test conditions): low
- Stability in water: Test item is hydrolyzed (half-life: within 30 min)
Analytical monitoring:
yes
Details on sampling:
- Sampling method: sampled directly by each vessel and pre-treatment was not performed
- Sample storage conditions before analysis: no strage before analysis
- Methanol was measured, since the test item was quickly hydrolyzed
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: test item was measured and diluted in dilution water by stirring for 120 min
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Alga
- Strain: ATCC22662
- Source (laboratory, culture collection): American Type Culture Collection
- Age of inoculum (at test initiation): after 4-day preculture
- Method of cultivation: on a shaker

ACCLIMATION
- Acclimation period: 4 days (2006-02-10 ~ 2006-02-14)
- Culturing media and conditions (same as test or not): same as test
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Test temperature:
23.0 - 23.1 °C (23 ± 2 °C), measured at the study initiation, 24, 48 and 72 hours after exposure
pH:
8.0 - 9.9, measured at the initiation and at the end of the study.
Nominal and measured concentrations:
Nominal concentration: 100 mg/L
Measured concentration: 89 mg/L (time-weighted mean; the concentration was determined based on the measured concentration of the hydrolysis product, methanol)
Details on test conditions:
TEST SYSTEM
- Test vessel: 500 mL Elenmyer-flask
- Type (delete if not applicable): closed
- Material, headspace, fill volume: glass, 490 mL, 100 mL
- Initial cells density: 5000 cells/mL
- Control end cells density: 855900 ± 20700 cells/mL
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Culture medium different from test medium: same medium

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no adjustment
- Photoperiod: continuously
- Light intensity and quality: 65 µE/m2/s

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: counting chamber; electronic particle counter;

TEST CONCENTRATIONS
- Range finding study: performed twice.
- Test concentrations: 1) 1, 10 and 100 mg/L, 2) 100 mg/L
- Results used to determine the conditions for the definitive study:
1) Inhibition rate was 16% (close system), -42% (open system) in 1 mg/L, 12% (close system), -44% (open system) in 10 mg/L, and 1% (close system), -36% (open system) in 100 mg/L, respectively.
2) Inhibition rate was -14% (close system) and 6% (open system). Measured concentration was 108 mg/L at the study initiation and 87 mg/L in a close system at the end of the study.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate (analytical grade)
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 89 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: the concentration was determined based on the measured concentration of the hydrolysis product, methanol
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 89 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: the concentration was determined based on the measured concentration of the hydrolysis product, methanol
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 89 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: the concentration was determined based on the measured concentration of the hydrolysis product, methanol
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 89 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: the concentration was determined based on the measured concentration of the hydrolysis product, methanol
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no abnormality was observed
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- EC50: 0.428 ± 0.0683 mg/L
Reported statistics and error estimates:
Student's t-test, subsequent to F test for homogeneity of variances.

Table 1. Calculated concentration of the test substance using methanol concentration

Nominal concentration

(mg/L)

Mean measured concentration, mg/L

(Percent of nominal)

Mean measured

concentration (*), mg/L

 

0 hour

24 hours

48 hours

72 hours

 

Control

< 0.03

< 0.03

< 0.03

< 0.03

 

100

105

(105

100

(100

75.3

(75)

80.2 (80)

89

(89)

*: Time-weighted mean measured concentration

Table 2. Growth inhibition (%)

Nominal conc. (mg/L)

[Mean measured conc. *]

Inhibition rate (%)

Growth rate

Inhibition rate (%)

Biomass

Control

-

-

100

[89]

0.3

0.5

*: Time-weighted mean measured concentration

Conclusions:
The NOEC of 2768-02-7 to algae was reported to be ≥89 mg/l for both biomass and growth rate. Due to the rapid rate of hydrolysis, the concentration was determined based on the measured concentration of the hydrolysis product, methanol.

Description of key information

Toxicity to algae: 70.5 hour EC50 >988 mg/l and NOELR: ≥988 mg/l (nominal) (highest concentration tested) (guideline ISO 10253 (Water quality - Marine Algal Growth Inhibition Test with Skeletonema costatum and Phaeodactylum tricornutum)).

Key value for chemical safety assessment

Additional information

A 70.5 hour EL50 of >988 mg/l and NOELR of ≥988 mg/l (nominal) (highest concentration tested) have been determined for the effects of the registration substance on growth rate of the marine algal species Skeletonema costatum, conducted in accordance with test guideline ISO 10253 and in compliance with GLP (TNO, 2000). These values are above the water solubility limit of the substance (0.0073 – 0.0088 mg/l at 23°C) and indicate that toxic effects on algae are unlikely to occur at this concentration. For monoconstituent substances it would not normally be appropriate to test at concentrations above the solubility limit.

This is the only reliable study available for this substance and it has been chosen as key.

The data above are supported by read-across evidence relevant to the impurities. The registration substance has an average purity of >70% Vi4-D4, with <20% 2,4,6,8,10-pentamethyl-2,4,6,8,10-pentavinylcyclopentasiloxane Vi5-D5 (CAS 17704-22-2; Impurity 1) and <10% 2,4,6-trimethyl-2,4,6-trivinylcyclotrisiloxane Vi3-D3 (CAS 3901-77-7; Impurity 2) present as impurities. After due consideration of the properties, the presence of these impurities is not expected to affect the overall hazard profile of the substance.

Read-across studies are in place as supporting studies, to consider the properties of the impurities. Data for Vi5-D5 are read-across from decamethylcyclopentasiloxane D5 (CAS 541-02-6), data for Vi3-D3 are read-across from hexamethyltrisiloxane D3 (CAS 541-05-9). These siloxanes have similar properties with regard to aquatic ecotoxicity.  Further information is given in a supporting report (PFA, 2017) attached in Section 13 of the IUCLID dossier.

A reliable algal effects study is available for the siloxane D5 (CAS 541-02-6) and is read-across to Impurity 1 (Vi5-D5) (Springborn Laboratories, 2001). This study (96 hour static test with Pseudokirchneriella subcapitata (formerly Selenastrum capricornutum) gave a result 96 hour EC50 >12 µg/l and 96 hour NOEC ≥12 µg/l. Effects were not seen within the limit of solubility in test media.

A reliable algal effects study is available for the siloxane D3 (CAS 541-05-9) and is read-across to Impurity 2 (Vi3-D3) (Springborn Smithers, 2007). This study (72 hour, static test with Pseudokirchneriella subcapitata) gave a result 72 hour EC50 >1.6 mg/l and 72 hour NOEC ≥1.6 mg/l. Effects were not seen within the limit of solubility in test media.

A 72 hour EC50 value of >89 mg/l (measured (TWA)) has been determined for the effects of read-across substance trimethox(vinyl)silane (CAS 2768-02-7) on growth rate of Pseudokirchneriella subcapitata (MOE, 2006). The data indicate that the vinyl side chain is unlikely to contribute significant excess ecotoxicity to the substance.