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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:
2007-02-06 to 2007-02-09
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Sampling method: At test initiation, 24, 48 and 72 hours, a single sample was removed from each test solution (i.e., 7.5, 15, 30, 60 and 120 mg a.i./L) and the control and analyzed for 3-(triethoxysilyl)propiononitrile.  Three quality control (QC) samples were prepared at test initiation and analyzed at each interval for 3-(triethoxysilyl)propiononitrile.  The QC samples were prepared at test concentrations similar to the treatment level range.  Results of these analyses indicated the accuracy of the analytical method for measuring the test substance concentration in the stock solution.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION

- Method: : A 120 mg a.i./L stock solution was prepared prior to test initiation by placing 0.2437 g of 3-(triethoxysilyl)propiononitrile (0.2400 g as active ingredient) in a 2000-mL volumetric flask and bringing it to volume with AAP medium.  The results stock solution was observed to be clear and colorless with a small amount of visible undissolved test substance.  The solution was mixed for approximately 40 minutes and was observed to be clear and colorless with no visible undissolved test substance after mixing.  Three of the test solutions (i.e., 15, 30 and 60 mg a.i./L) were prepared by bringing the appropriate amount of the 120 mg a.i./L stock solution (125, 250 and 500 mL) to a final volume of 1000 mL with AAP medium.  The 7.5 mg a.i./L test solution was prepared by bringing 125 mL of the 60 mg a.i./L solution to a final volume of 1000 mL with AAP medium.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM

- Strain: strain 1648

- Source: The alga was obtained from the University of Texas, Austin, Texas, and was maintained in stock culture at Springborn Smithers.  The stock cultures were maintained within the following conditions:  a shaking rate of 100 ± 10 rpm, a temperature of 23 ± 2  C and continuous illumination at the surface of the medium with an intensity of 4440 to 5900 lux (420 to 550 footcandles).  Lighting was supplied by fluorescent bulbs.  Culture flasks were agitated continuously on orbital shakers.

- Growt medium:  The culture medium used was Algal Assay Procedure (AAP) medium prepared with sterile, deionized water.  
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
Not reported
Test temperature:
24ºC
pH:
7.3 at test initiation.  At 72 hours of exposure, the pH of the test and control solutions ranged from 7.2 to 8.2.  
Dissolved oxygen:
No data
Salinity:
Not applicable
Nominal and measured concentrations:
Nominal concentrations: 0 (Control), 7.5, 15, 30, 60 and 120 mg/L.

Geometric mean measured concentrations:  0 (Control), 0.30, 0.52, 0.89, 1.8 and 3.6 mg/L.

The test results are presented and reported with reference to geometric mean measured concentrations.
Details on test conditions:
TEST SYSTEM

- Exposure vessel type: The test was conducted in sterile 250-mL Erlenmeyer flasks fitted with stainless steel caps.

- Test Design:  One hundred milliliters of the appropriate exposure solution was placed in each replicate vessel.  A 0.686-mL inoculum of Pseudokirchneriella subcapitata cells, at a density of approximately 145.7 x 104 cells/mL, was aseptically introduced into each vial.  This inoculum provided an initial (0 hour) cell density of approximately 1.0 x 104 cells/mL.  Three replicate test vessels were established for the treatment levels and six replicates were established for the control.  

- Growth/test medium:  AAP medium used to prepare the exposure solutions was formulated in the same manner as the culture medium. TOC concentration of the AAP sample collected in February 2007 was 0.53 mg/L. 

- Light levels and quality during exposure:  4500 to 5500 lux (420 to 510 footcandles).  The photosynthetically-active radiation (PAR) of the test area measured at test initiation ranged from 68 to 84 µE/m2/s.
Reference substance (positive control):
no
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
1.8 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: hydrolysis product also present in solution
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 3.6 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: hydrolysis product also present in solution
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 3.6 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: hydrolysis product also present in solution
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 120 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: hydrolysis product also present in solution
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
60 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: hydrolysis product also present in solution
Details on results:
- Exponential growth in the control (for algal test): yes
Reported statistics and error estimates:
Based on the results of Shapiro-Wilks' and Bartlett's Tests, this data set passed the requirements for normality and homogeneity of variance, therefore, Williams' Test was used to determine treatment-related effects.  Since no concentration tested resulted in >=50% reduction of yield, the 72 hour ErC50 and EyC50 values were empirically estimated to be >the highest geometric mean measured concentration tested.

Table 1. Test results

 Geometric mean measured concentration (mg/L)  Mean cell density after 24 hours (cells/ml)   Mean cell density after 48 hours (cells/ml)   Mean cell density after 72 hours (cells/ml)
 0 (Control)  56300  185000  1240000
 0.30  56700  196000  1250000
 0.52  50800  199000  1250000
 0.89  43300  218000  1170000
 1.8  36700  181000  1090000
 3.6  35800  140000  940000

Yield
The 0- to 72-hour yield in the control averaged 122.93 x 104
cells/mL.  The 0- to 72-hour yield in the 0.30, 0.52, 0.89,
1.8 and 3.6 mg/L treatment levels averaged 124.00, 124.33,
116.25, 107.75 and 93.42 x 104 cells/mL, respectively. 
Based on the results of Shapiro-Wilks' and Bartlett's Tests,
this data set passed the requirements for normality and
homogeneity of variance, therefore, Williams' Test was used
to determine treatment-related effects.  Williams' Test
determined a significant reduction in yield occurred in the
3.6 mg/L treatment level compared to the control data. 
Therefore, the 72 hour NOEC for yield was determined to be
1.8 mg/L.  The 72 hour EyC10 was determined to be 1.4 mg/L,
with 95% confidence intervals of 0.49 and 2.7 mg/L.  The
72-hour EyC20 was determined to be 2.5 mg/L, with 95%
confidence intervals of 0.88 and 3.5 mg/L.  Since no
concentration tested resulted in >=50% reduction of yield,
the 72 hour EyC50 value was empirically estimated to be >
3.6 mg/L, the highest geometric mean measured concentration
tested.

Growth Rate
The 0- to 72-hour growth rate in the control averaged 1.65
days-1.  The 0- to 72 hour growth rate in the 0.30, 0.52,
0.89, 1.8 and 3.6 mg a.i./L treatment levels averaged 1.65,
1.66, 1.63, 1.61 and 1.56 days 1, respectively.  Based on
the results of Shapiro-Wilks' and Bartlett's Tests, this
data set passed the requirements for normality and
homogeneity of variance, therefore, Williams' Test was used
to determine treatment-related effects.  Williams' Test
determined a significant reduction in average growth rate
occurred in the 3.6 mg a.i./L treatment level compared to
the control data.  Based on these results, the 72 hour NOEC
was determined to be 1.8 mg a.i./L.  Since no concentration
tested resulted in >= 10% reduction in growth rate, the 72
hour ErC10, ErC20 and ErC50 values were empirically
estimated to be > 3.6 mg a.i./L, the highest geometric mean
measured concentration tested.

Element value (e.g. ErC50 and EyC50): 
72-hour EyC10:  1.4 mg/L
72-hour EyC20:  2.5 mg/L
72-hour EyC50:  > 3.6 mg/L
72-hour ErC10:  > 3.6 mg/L
72-hour ErC20:  > 3.6 mg/L
72-hour ErC50:  > 3.6 mg/L

NOEC: 1.8 mg/L (for growth rate (r) and biomass expressed as yield(y))

Validity criteria fulfilled:
yes
Conclusions:
A 72-hour EC50 value of >3.6 mg/L and NOEC of 1.8 mg/L have been determined for the effects of the test substance on growth rate of Pseudokirchnerella subcapitata. The test results are expressed relative to the concentration of the test substance however it is likely that the test organisms were exposed to a mixture of the test substance and its hydrolysis products. The EC50 and NOEC values expressed relative to nominal test concentration are >120 and 60 mg/L respectively.

Description of key information

72 h ErC50 >74 mg/l and NOEC 37 mg/l, growth rate P. subcapitata, read-across from CAS 919-31-3, expressed in terms of concentration of the silanol hydrolysis product 3-(trihydroxysilyl)propiononitrile.

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:
37 mg/L

Additional information

There are no reliable algal toxicity data available for 3-(trimethoxysilyl)propiononitrile (CAS 2526-62-7), therefore good quality data for an appropriate structural analogue, 3-(triethoxysilyl)propiononitrile (CAS 919-31-3), have been read across. Both substances share the same silanol hydrolysis product, 3-(trihydroxysilyl)propiononitrile. The other hydrolysis products are methanol and ethanol, respectively. In the test with 3-(triethoxysilyl)propiononitrile (CAS 919-31-3), the observations are attributed to the exposure of test organisms to a mixture of the parent substance, 3-(triethoxysilyl)propiononitrile, and the silanol hydrolysis product, 3-(trihydroxysilyl)propiononitrile in the test system. There is no basis to expect that ethanol significantly influenced the results of the test. The toxicity of ethanol is discussed further in the ecotoxicological information overview endpoint summary (additional information).

 

A 72-h ErC50 value of >120 mg/l, ErC10 value of >120 mg/l and NOEC of 60 mg/l (nominal concentration) have been determined for the effects of 3-(triethoxysilyl)propiononitrile (CAS 919-31-3) on growth rate of Pseudokirchneriella subcapitata in accordance with test guideline OECD 201 (Springborn Smithers, 2007). In view of the test media preparation method and static exposure regime it is likely that the test organisms were exposed to a mixture of the parent substance and the hydrolysis products of the tested substance. The study was supported by GC-FID analysis of the parent substance and EC50 and NOEC values in terms of concentrations of parent were >3.6 mg/l and 1.8 mg/l respectively, consistent with the expectation of degradation due to hydrolysis in the test media. The results may be expressed in terms of concentration of the hydrolysis product, 3-(trihydroxysilyl)propiononitrile, by applying a molecular weight correction to the nominal effect concentration: (MW of silanol = 133.18 / MW of parent = 217.34) * [CONCENTRATION OF PARENT: EC50 >120 mg/l and NOEC 60 mg/l] = EC50 >74 mg/l and NOEC 37 mg/l. These values are read across to 3-(trihydroxysilyl)propiononitrile without further correction.

Effects of the hydrolysis product 3-(trihydroxysilyl)propiononitrile on growth rate of algae are not expected, as the QSAR predicted value implies.

The hydrolysis half-life of 3-(triethoxysilyl)propiononitrile (CAS 919-31-3) is approximately 6.5 hours, so it is expected that the algae were exposed to the parent test substance at the beginning of the test, within the first 0-24 hour timepoint. When looking at the reported cell density values at 0-24 hours, the cell density increase in the control was by a factor of approximately 5.63 cells/ml, whereas in the highest test concentration (120 mg/l nominal) the increase was by a factor of approximately 3.58 cells/ml. At 24-48 hours the control cell density increased by a factor of approximately 3.28 cells/ml while the 120 mg/l concentration increased by a factor of approximately 3.9 cells/ml. And then, at 48-72 hours, the control cell density increased by a factor of approximately 6.7 cells/ml while the 120 mg/l concentration also increased by a factor of approximately 6.7 cells/ml. This raw data clearly shows that cell density in the highest test concentration increased less than the cell density in the control during the first 24 hours of the test, but that cell density increase at the 24-48 hour and 48-72 hour timepoints were no different to the control.

 

The 72 hour NOEC value of 60 mg/l is therefore attributed to exposure of the algae to the parent substance, 3-(triethoxysilyl)propiononitrile (CAS 919-31-3). Because the hydrolysis half-life of the registration substance is faster than that of the read-across substance, it is not expected that organisms would be significantly exposed to the parent registration substance. The algal NOEC, read-across from 3-(triethoxysilyl)propiononitrile (CAS 919-31-3), is therefore very conservative.

The read-across is supported by an algal 96 h EC50 QSAR prediction value of 110000 mg/l for the silanol hydrolysis product, 3-(trihydroxysilyl)propiononitrile, using ECOSAR v2.0 for the prediction of the aquatic toxicity of neutral organics.