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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:
2004-05-06 to 2004-05-09
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study with GLP but no analysis of exposure concentrations
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
GLP compliance:
yes
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION

- Method: A 100 mg a.i./L. (mg active ingredient/L) stock solution was prepared by adding 215 µL of trimethoxysilane (0.1998 g based on a purity of 97.1% and a density of 0.957 g/mL) to 2000 mL of dilution water. The solution was mixed overnight with a magnetic stir plate and Teflon-coated stir bar. Each test concentration was prepared by adding the appropriate amount of the 100 mg a.i./L stock solution to an intermediate vessel and bringing it to a final volume of 1000 mL with dilution water.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM

- Strain: Psuedokirchneriel/a  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 Laboratories. 

- Method of cultivation: The stock cultures were maintained within the following conditions: 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 6300 to 9600 lux (590 to 890 foot candles). Lighting was supplied by fluorescent bulbs. Culture flasks were agitated continuously on an orbital shaker. 


ACCLIMATION

- Culturing media and conditions (same as test or not): no
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
No data
Test temperature:
24°C
pH:
7.0 to 7.1 at start of test

8.6 to 9.3 at end of test
Dissolved oxygen:
No data
Salinity:
Not applicable
Nominal and measured concentrations:
Nominal concentrations: 0 (Control), 6.3, 13, 25, 50 and 100 mg/L
Details on test conditions:
TEST SYSTEM

- Test vessel: The test was conducted in sterile 250-mL Erlenmeyer flasks containing 100 mL of test solution. All test vessels were fitted with stainless steel caps which permitted gas exchange.

- Test Design: One hundred milliliters of the appropriate exposure solution was placed in each replicate flask. A 0.226mL inoculum of Psuedokirchneriella subcapitata cells, at a density of approximately 443 x 10(4) cells/mL, was aseptically introduced into each flask. This inoculum provided the required initial (0 hour) cell density of approximately 1.0 x 10(4)cells/mL. Three replicate test vessels were established for the treatment levels and the dilution water control.

- Water chemistry in test: TOC concentration of the AAP sample collected in May 2004 was 0.46 mg/L. Conductivity of the exposure and control solutions measured at test initiation and termination was maintained at 80 umhos/cm.  

GROWTH MEDIUM

- Standard medium used: yes

- Detailed composition if non-standard medium was used: 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.


OTHER TEST CONDITIONS

- Light intensity and quality: 6500 to 8600 lux (600 to 800 footcandles). The photosynthetically-active radiation (PAR) of the test area measured at test initiation ranged from 98 to 128 µE/m2/s.

TEST CONCENTRATIONS

- Spacing factor for test concentrations: 2

- Range finding study

- Test concentrations: 0.01, 0.1, 1. 10 and 100 mg/L

- Results used to determine the conditions for the definitive study: No significant difference in cell densities compared with the Control after 72 hours
Reference substance (positive control):
no
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
< 6.3 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: growth rate and biomass
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: growth rate and biomass
Details on results:
After 72 hours of exposure, cells exposed to all the treatment levels tested and the control were observed to be normal.  The 72 hour cell density in the control averaged 154.00 x 10 (4) cells/mL. Cell densities in the 6.3, 13, 25, 50 and 100 mg a.i./L treatment levels averaged 104.75, 117.58, 104.33, 117.72 and 119.14 x 10(4) cells/mL, respectively.
Reported statistics and error estimates:
Average-specific growth rates and Areas under the growth curves were determined for each treatment level in accordance with OECD guidance. No test concentration resulted in >50% effect on growth and therefore the EC50 values were estimated empirically from the data as being >than the highest treatment.

After checking for homogeneity of variance with Bartlett's test and Shapiro-Wilks' test, the NOECs were determined by Williams' test at p≤5%.

Table 1. Test results

 Nominal test substance concentration (mg/L)  Initial cell concentration (cells/mL) (SD)  Mean cell concentration after 24 hours (cells/mL) (SD)   Mean cell concentration after 48 hours  (cells/mL) (SD)  Mean cell concentration after 72 hours  (cells/mL) (SD)  
 0 (Control)  10000  47500 (18900)  280000 (47500)  1540000 (237500)
 6.3  10000  47500 (4300)  167500 (25400)  1047500 (218800)
 13  10000  52500 (2500)  160000 (10900)  1175800 (143700)
 25  10000  40000 (10000)  181700 (36400)  1043300 (110000)
 50  10000  38300 (15900)  200000 (26500)  1177200 (206900)
 100  10000  43300 (22700)  127500 (69300)  1191400 (231900)

Biomass:  The total biomass in the control averaged 101.37 x 10(4) cells-days/mL. Total biomass in the 6.3, 13, 25, 50 and 100 mg a.i./L treatment levels averaged 67.53, 73.38, 67.92, 75.81 and 70.15 x 10(4) cells-days/mL, respectively.  Williams' Test determined a significant difference in the control (101.37 x 10(4) cells-days/mL). The NOEC for total biomass was determined to be <6.3 mg a.i./L, the lowest nominal concentration tested.  Since no concentration tested resulted in ≥50% inhibition, the 72-hour EbC50 was empirically estimated to be >100 mg a.i./L, the highest nominal concentration tested. Growth rate:  The 0 to 72 hour growth rate in the control averaged 1.72 days-1.  The 0-72 hour growth rate in the 6.3, 13, 25, 50 and 100 mg a.i./L treatment levels averaged 1.58, 1.63, 1.59, 1.63 and 1.63 days-1, respectively.  Statistical analysis (Williams' Test) determined a significant reduction in all treatment levels tested when compared to the growth rate in the control (1.72 days-1).  The 72 hour NOEC for growth rate was determined to be <6.3 mg a.i./L, the lowest nominal concentration tested.  Since no concentration tested resulted in ≥50% inhibition, the 72-hour EC50 was empirically estimated to be >100 mga.i./L, the highest nominal concentration tested.

Validity criteria fulfilled:
yes
Conclusions:
A 72-hour EC50 of >100 mg/L and a NOEC of <6.3 mg/L have been determined for the effects of the test substance on growth rate and biomass of Pseudokirchnerella subcapitata. It is likely that the test organisms were 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:
2006-08-15 to 2006-08-18
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
Analytical monitoring:  A single sample was removed from each test solution and the control at test initiation and test termination (72 hours) and was extracted and analyzed by GC/FID for ethyl silicate concentration.  Samples analyzed on day 0 were removed from the test solutions in the volumetric flasks prior to filling the individual test flasks.  Samples analyzed at 72 hours of exposure were removed from the composite of replicate vessels for each treatment and the control.  
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION

- Method: A 100 mg a.i./L (mg active ingredient/L) stock solution was prepared by placing 0.2025 g of ethyl silicate (0.2005 g as active ingredient) in a 2000-mL volumetric flask and bringing it to volume with AAP medium.  The resulting stock solution was observed to be clear and colorless with no visible undissolved test substance.  Each test concentration was prepared by adding the appropriate amount of the 100 mg a.i./L stock solution to an intermediate vessel and bringing it to a final volume of 1000 mL with dilution water.
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: The alga was obtained from the University of Texas, Austin, Texas, and was maintained in stock culture at Springborn Smithers.  

- Culture conditions: The stock cultures were maintained within the following conditions:  a shaking rate of 100 ± 10 rpm, a temperature of 23 ± 1ºC and continuous illumination at the surface of the medium with an intensity of 7000 to 9100 lux (650 to 800 footcandles).  Lighting was supplied by fluorescent bulbs.  Culture flasks were agitated continuously on an orbital shaker.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
Not reported
Test temperature:
23-24ºC
pH:
The pH of the test and control solutions ranged from 6.7 to 6.8 at test initiation.  At 72 hours of exposure, the pH of the test and control solutions ranged from 7.1 to 8.5. 
Dissolved oxygen:
Not reported
Salinity:
Not applicable
Nominal and measured concentrations:
Nominal concentrations: 0(Control), 2.6, 6.4, 16, 40 and 100 mg a.i./L 

Measured (geometric mean) concentrations: 0(Control), 1.8, 0.92, 3.6, 8.6 and 22 mg a.i./L
Details on test conditions:
- 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.

- Exposure vessel type: The test was conducted in sterile 250-mL Erlenmeyer flasks containing 100 mL of test solution.  All test vessels were fitted with stainless steel caps which permitted gas exchange.  

- Water chemistry in test:  TOC concentration of the AAP sample collected in August 2006 was 0.51 mg/L. 

- Conductivity of the exposure and control solutions measured at test initiation and termination ranged from 80 to 90 µmhos/cm.  

- Light levels and quality during exposure:  7000 to 9100 lux (650 to 850 footcandles).  The photosynthetically-active radiation (PAR) of the test area measured at test initiation ranged from 90 to 140 µE/m2/s.

- Test Design:  One hundred milliliters of the appropriate exposure solution was placed in each replicate flask.  A 0.23-mL inoculum of Pseudokirchneriella subcapitata cells, at a density of approximately 445 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.  Three replicate test vessels were established for the treatment levels and six replicates were established for the dilution water control.

- Method of calculating mean measured concentrations (i.e. arithmetic mean, geometric mean, etc.): Geometric mean
Reference substance (positive control):
no
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: growth rate and biomass
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: growth rate and biomass
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 22 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: growth rate and biomass
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 22 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: growth rate and biomass
Details on results:
- Exponential growth in the control (for algal test): yes
Reported statistics and error estimates:
Statistical methods: The data were first checked for normality using Shapiro-Wilks' Test (Weber et al., 1989) and for homogeneity of variance using Bartlett's Test (Horning and Weber, 1985).  If the data sets passed the tests for homogeneity and normality, then Williams' Test (Williams, 1971, 1972) was used to determine the NOEC.  If the data did not pass the tests for homogeneity and normality, then Kruskal-Wallis' Test (Sokal and Rohlf, 1981) was used to determine the NOEC.  All statistical determinations were made at the 95% level of certainty, except in the case of Shapiro-Wilks' and Bartlett's Tests, where the 99% level of certainty was applied.  TOXSTAT® version 3.5  (Gulley et al., 1996) was used to perform these calculations.

Table 1. Test results

 Nominal test substance concentration (mg/L)  Geometric mean measured concentration (mg/L)  Mean measured cell concentration at start of test (cells/mL)   Mean measured cell concentration after 24 hours (cells/mL)      Mean measured cell concentration after 48 hours (cells/mL)   Mean measured cell concentration after 72 hours (cells/mL)  
 0 (Control)  -  10000  34600  261300  1030100
 2.6 1.8  10000  40000  288300  1236900
 6.4 0.92  10000  35000  314200 930000 
 16 3.6  10000  45800  365000  1343600
 40 8.6  10000  40800  289200  1677800
 100 22   10000  48300  287500  1685600

Observations: After 72 hours of exposure, cells exposed to all treatment levels tested and the control were observed to be normal. 

Table 2. Test results

 Nominal test substance concentration (mg/L)  Geometric mean measured concentration (mg/L)   Yield (0 -72 hours) x 10000 cells/ml  Growth rate (0 -72 hours)  Inhibition of yield (biomass) at end of test (%)  Inhibition of growth rate at end of test (%) 
 0 (Control)  -  102  1.52  -  -
 2.6 1.8  123 1.59  -20  -5
 6.4 0.92 92 1.49   10  2
 16 3.6  133  1.61  -31  -6
 40 8.6  167  1.68  -63  -11
 100 22   168  1.69  -64  -11

 

Validity criteria fulfilled:
yes
Conclusions:
A 72-hour EC50 value of >22 mg/L and NOEC of ≥22 mg/L have been determined for the effects of the test substance on growth rate and biomass of Pseudokirchnerella subcapitata based on geometric mean measured concentrations (>100 mg/L and ≥100 mg/L respectively based on nominal concentration). It is likely that the test organisms were exposed to the hydrolysis products of the substance.

Description of key information

Toxicity to algae: 72-hour ErC50: >100 mg/l and NOEC: <6.3 mg/l, growth rate of Pseudokirchneriella subcapitata; equivalent to ErC50: >79 mg/l and NOEC <5 mg/l as monosilicic acid, read across from trimethoxysilane (CAS 2487-90-3).

Key value for chemical safety assessment

Additional information

There are no reliable short-term data available for trichlorosilane (CAS 10025-78-2) therefore good quality data from an appropriate structural analogue, trimethoxysilane (CAS 2487-90-3), have been read across. The substances share the same silanol hydrolysis product, monosilicic acid. The other hydrolysis products are hydrogen chloride for trichlorosilane (CAS 10025-78-2) and methanol for trimethoxysilane (CAS 2487-90-3).

A 72-hour ErC50 value of >100 mg/l and NOEC value of <6.3 mg/l (nominal concentration) have been determined for the effects of trimethoxysilane (CAS 2487-90-3) on growth rate of the freshwater algae Pseudokirchneriella subcapitata, in accordance with OECD 201 and in compliance with GLP. (Springborn Smithers, 2004c).

The test substance is susceptible to hydrolysis and, due to the test media preparation (stirring overnight) and exposure regime (static), it is likely that the test organisms were predominantly exposed to the hydrolysis products of the substance, monosilicic acid and methanol. 

The results may be expressed in terms of concentration of the hydrolysis product, monosilicic acid, by applying a molecular weight correction: (MW of silanol = 96.10 / MW of parent = 122.20) * ErC50 >100 mg/l and NOEC <6.3 mg/l = ErC50 >79 mg/l and NOEC <5 mg/l.  

 

Supporting data have also been read across from tetraethyl orthosilicate (CAS 78-10-4), which hydrolyses rapidly to monosilicic acid and ethanol:

A 72-hour EC50 value of >100 mg/l and NOEC of ≥100 mg/l (nominal concentration) (highest concentration tested) have been determined for the effects of tetraethyl orthosilicate (CAS 78-10-4) on growth rate and biomass of Pseudokirchneriella subcapitata (Springborn Smithers, 2008) in accordance with test guideline OECD 203 and in compliance with GLP.

In view of the test media preparation method/exposure regime it is likely that the test organisms were exposed predominantly to the hydrolysis products of the tested substance. Measured concentrations of the parent substance were determined at the beginning and end of the test. However, in view of the rapid hydrolysis rate of the substance, the measured concentrations of the parent substance are not thought to be relevant. The effect concentrations reported are therefore expressed in terms of nominal concentration, corrected to concentration of the silicon hydrolysis product by means of a molecular weight correction.

The results may therefore be expressed in terms of concentration of the hydrolysis product, monosilicic acid, by applying the following molecular weight correction: EC50: (MW of silanol = 96.1 / MW of parent = 208.33) * >100 = >46 mg/l; NOEC: (MW of silanol = 96.1 / MW of parent = 122.2) * ≥100 = ≥46 mg/l. 

 

Refer to IUCLID Section 6, CSR Section 7, and the ecotoxicity RAAF report attached in IUCLID Section 13 or as an Annex in the CSR, for further discussion of the approach to chemical safety assessment and justification for read across.