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Toxicity to aquatic algae and cyanobacteria

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Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
calculation (if not (Q)SAR)
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The result was obtained by an appropriate predictive method.
Principles of method if other than guideline:
The ECOSAR ‘neutral organics’ QSARs for acute data have been applied and the effect concentrations calculated using log Kow and molar mass as input variables. An additional factor of *0.2 has been applied to the results.
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
120 mg/L
Basis for effect:
growth rate
Conclusions:
A 96 h EC50 values of 120 mg/L was obtained for the hydrolysis product of the submission substance using an appropriate calculation method . The results are considered to be reliable.
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2009-09-29 to 2009-10-02
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 (incl. certificate)
Analytical monitoring:
no
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION

- Method: Prior to test solution preparation, a dosing stock solution was prepared by mixing 1.52 mL of Trichloro(phenyl)silane (corresponding to 2 g) with 2 mL of tetrahydrofuran (THF) using Hamilton syringes. The solution was shaken by hand. A 100 mg test item/L stock solution
was prepared prior to test initiation by adding 176 μL of the dosing stock solution to 800 mL algal medium using a Hamilton syringe. Prior to addition of the dosing stock solution, the measurement flask containing the algal medium was placed on a magnetic stirrer. The spiked solution was stirred continuously over night. The pH of the solution was then adjusted to 7.0 with 0.1 N sodium hydroxide (NaOH). Thereafter, the stock solution was further diluted to a final volume of 1000 mL with algal medium, resulting in a solvent (THF) concentration of 0.10 mL/L. Nominal test concentrations of 6.25, 12.5, 25, 50 and 100 mg/L were prepared by dilution of the stock solution and addition of THF to a final concentration of 0.1 mL/L. All resulting test solutions were shaken by hand for 30 seconds and observed to be clear and colorless, with no visible undissolved test item.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM

- Source (laboratory, culture collection): The alga was originally obtained from Albrecht-v.-Haller-Institut, Göttingen, Germany, and was maintained in stock culture at Springborn Smithers Laboratories (Europe).

- Culture medium: The culture medium used was a synthetic algal assay growth medium (OECD, 2006), prepared by adding appropriate amounts of nutrient stock solutions to sterile, deionized water. Representative samples of the dilution water source used in the preparation of the culture medium were analyzed periodically for the presence of pesticides, PCBs and toxic metals conducted at Bachema, Schlieren, and Interlabor Belp AG, Belp, Switzerland. None of these compounds have been detected at concentrations that are considered toxic to algae in any of the water samples analyzed in agreement with ASTM guidelines.

- Preparation of starter culture: Four days prior to test start, the culture was maintained within the following conditions: a shaking rate of 120 rpm, a temperature of 23ºC and continuous illumination of 7091 to 7123 lux. Lighting was supplied by fluorescent bulbs. Culture flasks were shaken
continuously on an orbital shaker. Temperature was controlled using an environmental chamber. The inoculum used to initiate the toxicity test was taken from a stock culture that had been transferred to fresh medium four days before testing.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
Not reported
Test temperature:
23.4 to 23.7ºC
pH:
7.30 to 7.65 at test initiation

6.08 to 9.86 at end of test
Dissolved oxygen:
Not reported
Salinity:
Not applicable
Nominal and measured concentrations:
Nominal test concentrations: 0 (Control), 0 (Solvent control), 6.25, 12.5, 25.0, 50.0 and 100 mg test item/L
Details on test conditions:
TEST SYSTEM

Test vessels: Three sterile 250-mL Erlenmeyer flasks (A to C) for the controls and the treatment levels were prepared. An aliquot of 100 mL of the appropriate test solution was placed in each replicate flask. All test vessels were fitted with stainless steel caps which permitted gas exchange.

- Inoculum: An aliquot of 0.25 mL of the preculture of Pseudokirchneriella subcapitata, at a density of 394 x 104 cells/mL, was then inoculated aseptically in each 250 mL volumetric flask. The resulting slight dilution from the inoculum was considered to be negligible. These volumes of inocula provided the required cell density of 1.0 x 104 cells/mL.

- Algal growth medium: The algal medium used to prepare the exposure solutions was the same as the culture medium. The medium was equilibrated to test temperature. The pH of the medium was 8.03.

- Test conditions: The test was conducted in a temperature-controlled room to maintain the test conditions specified in the study plan: a temperature of 24 ± 2ºC and continuous light intensity of 4400 to 8800 lux. An orbital shaker table provided a shaking rate of 100 rpm. Test flasks were randomly placed on the shaker table at test initiation. Following each observation interval the test flasks were assigned new random positions.

- Algal growth measurement: At each subsequent 24-hour interval, cell counts were conducted on each replicate vessel of the treatment levels and the control using a hemocytometer (Neubauer Improved) and a Leica DMLS microscope. One sample was removed from each flask for counting. One or more hemocytometer field(s), each 0.1 cm x 0.1 cm in size and 0.01 cm deep, containing 0.0001 mL of culture, were examined for each sample until at least 400 cells or a total of four fields were counted. Observations of the health of the algal cells were also made at each 24- hour interval.

- Monitoring culture conditions: Temperature was measured continuously with a minimum/maximum thermometer located in a flask of water adjacent to the test flasks in the environmental chamber. Minimum and maximum temperatures and the shaking rate of the orbital shakers (Gerhardt, 450625 R05) were recorded daily. Light intensity was measured with a Pancontrol LX 1308 at hour 0 and at each 24-hour interval during the exposure period. The pH of the test solutions and control was measured at test initiation and at the termination of the 72-hour exposure period. Test solution pH was measured using a Metrohm 691 pH-meter.
Reference substance (positive control):
no
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Details on results:
- Exponential growth in the control (for algal test): yes
Reported statistics and error estimates:
Definitive EC50 values could not be determined because they were above the highest treatment level.

ANOVA and William’s Test were used to determine the NOEC and LOEC values

Table 1. Test results

 Nominal test concentration (mg/L)  Initial cell density (cells/mL)  Mean cell density after 24 hours (cell/mL)   Mean cell density after 48 hours (cell/mL)  Mean cell density after 72 hours (cell/mL)  Perecntage inhibition in cell density*  Percentage inhibition in growth rate* Percentage inhibition in biomass*
 0 (Control)  10000  48000  247000  1070000  9.0  1.4  9.0
 0 (Solvent control)  10000  59000  228000  1180000  -  -  -
 6.25  10000  65000  279000  1240000  -5.3  -1.9  -5.4
 12.5  10000  49000  254000  1140000  2.7  -0.03  2.7
 25.0  10000  43000  240000  1050000  10.5  1.9  10.6
 50.0  10000 33000   203000  1030000  12.6  2.0  12.7
 100  10000  45000  248000  1030000  12.1  2.2  12.2

*Relative to solvent control. A negative (-) value indicates that growth was higher than in the solvent control.

Validity criteria fulfilled:
yes
Conclusions:
A 72-hour EC50 value of >100 mg/L and a NOEC of ≥100 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 primarily exposed to the hydrolysis products of the substance.
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
Please refer to the attached justification for grouping of substances provided in IUCLID Section 13.
Reason / purpose:
read-across source
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2008-05-27 to 2008-05-30
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Low test item concentrations used.
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0 (Control), 0 (Solvent control), 0.013, 0.025, 0.050, 0.10 and 0.20 mg/L

- Sampling: Since the test substance was expected to hydrolyze rapidly in the test solutions, (e.g., 0.4 hour half-life) analytical sampling was performed at test initiation and after 24 hours of exposure. At test initiation and at 24 hours of exposure, samples were removed from each test solution and the controls and analyzed for trimethoxyphenylsilane. Samples analyzed on day 0 were removed from the test solutions in the volumetric flasks prior to inoculation with algae and filling the individual replicate test flasks. Samples analyzed at 24 hours of exposure were removed from the approximate midpoint of the replicate test vessels and composited by treatment or control.

At test initiation and 24 hours of exposure, a sample was removed from the replicate flask (D) of the 0.050 mg a.i./L test concentration which did not contain algae. The result of these analyses were compared with that obtained for the 0- and 24-hour analysis of the 0.050 mg a.i./L solution containing algae to assess the impact that algae had on the test substance concentration.

Three quality control (QC) samples were prepared at each sampling interval at nominal concentrations approximating the test concentration range and remained with the exposure solution samples throughout the analytical process. The results of the QC sample analysis were used to judge the precision and quality control maintained during the analytical process.
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION

- Concentrations: Based on the limited water solubility of the test substance, the reported half-life of 0.4 hours at pH 7.0 and 25 ºC, the results of the preliminary range finding test and in consultation with the Study Sponsor, nominal concentrations of 0 (control and solvent control), 0.013, 0.025, 0.050, 0.10 and 0.20 mg a.i./L were selected for the definitive exposure.

- Method: A 2.0 mg a.i./mL stock solution was prepared by placing 0.0506 g of the test substance (0.0500 g as active ingredient) in a 25-mL volumetric flask and bringing it to volume with dimethylformamide (DMF, CAS No. 68-12-2). The resulting stock solution was observed to be clear and colorless with no visible undissolved test substance. This stock solution was used to prepare secondary stock solutions and the exposure solutions by dilution. The test solutions were prepared in volumetric flasks and due to the known rapid hydrolysis of the test substance, mixed for only one minute. Analytical samples (30.0 mL) were collected from the volumetric flasks containing the treatment, control and solvent control solutions. The solvent control solution was prepared by bringing 0.10 mL of DMF to a final volume of 1000 mL with AAP medium.

- Controls: AAP medium control and AAP medium + Dimethylformamide control

- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): 0.1 mL/L

- Evidence of undissolved material (e.g. precipitate, surface film, etc): All test solutions were clear and colorless with no visible undissolved test substance
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM

- Strain: strain 1648

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

- Culture medium: The culture medium used was Algal Assay Procedure (AAP) medium prepared with sterile, deionized water. If necessary, the pH of the culture medium was adjusted to pH 7.5 ± 0.1 with either 0.1 N hydrochloric acid or 1.0 N sodium hydroxide.

- Culture conditions: Stock cultures were grown in 250-mL glass flasks each containing 100 mL of medium. The flasks were covered with stainless steel caps which permitted gas exchange. The stock cultures were maintained within the following conditions: a shaking rate of 100 ± 10 rpm, a temperature of 23 ± 1 ºC and continuous illumination at the surface of the medium with an intensity range of 4400 to 5900 lux (420 to 550 footcandles). Lighting was supplied by Premira VitaLux® fluorescent bulbs. Culture flasks were agitated continuously on an orbital shaker. Temperature was controlled using an environmental chamber.

- Test inoculum: The inoculum used to initiate the toxicity test with trimethoxyphenylsilane was taken from a stock culture that had been transferred to fresh medium four days before testing.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
Not reported
Test temperature:
23-24°C
pH:
7.0-9.5
Dissolved oxygen:
Not determined
Salinity:
Not applicable
Nominal and measured concentrations:
Nominal concentrations: 0 (Control), 0 (Solvent control), 0.013, 0.025, 0.050, 0.10 and 0.20 mg/L..

At test initiation, measured concentrations established a concentration gradient and ranged from 64 to 90% of nominal concentrations. At 24 hours of exposure, measured concentrations in all the treatment levels had decreased to below the limit of quantitation for each sample. Since the 24-hour measured concentrations were below the limit of quantitation, no additional analytical samples were collected or analyzed.

The test substance rapidly hydrolyses and it is therefore likely that its hydrolysis products are still present in the test media. The concentrations of the hydrolysis products were not measured and the test results are therefore interpreted with reference to nominal test substance concentrations.
Details on test conditions:
- Inoculation of test media: To minimize the time between the addition of test substance to algal medium, and the inoculation with algal cells, the alga was added directly to the test solutions in the 1000 mL volumetric flasks, after the analytical samples were collected. Approximately ten minutes following preparation of the test solutions and analytical sampling (970 mL/flask), 7.3 mL of an inoculum of Pseudokirchneriella subcapitata cells, at a density of 132.69 x 10E4 cells/mL, was aseptically introduced into each volumetric flask containing 970 mL of test or control solution. This inoculum provided a cell density of approximately 1.0 x 10E4 cells/mL. The test solutions were then divided into separate replicate test flasks (100 mL/test flask).

- Test vessels: Replicate 250-mL test flasks, three per treatment level and the control and six for the solvent control, were conditioned prior to use by rinsing with the appropriate test solution. One hundred milliliters of the appropriate test solution was then placed in each replicate flask. Each test flask was labeled with the test concentration, replicate, test species and study number. All test vessels were fitted with stainless steel caps which permitted gas exchange.

In order to estimate the impact that the presence of algal biomass had on the test substance concentration, an additional replicate flask (D) of the 0.050 mg a.i./L (nominal) treatment level was prepared. This flask, which was not inoculated with algae, was analyzed at 0 and 24 hours of exposure for trimethoxyphenylsilane concentration. The results of the 24-hour analysis were compared with the 24-hour results for the 0.050 mg a.i./L solution containing algae.

Test flasks were randomly placed on the shaking table at test initiation based on computer-generated random numbers. Following each observation interval, the test flasks were assigned new random positions based on computer-generated random numbers.

- Test conditions: The test was conducted in an environmental chamber designed to maintain the test conditions specified in the protocol: a temperature of 23 ± 2°C, continuous light intensity of 4400 to 5900 lux (420 to 550 footcandles) and photosynthetically-active radiation (PAR).

- Observations and measurements: At each subsequent 24-hour interval, a single cell count was conducted on each replicate solution of the treatment level and the controls using a hemacytometer (Neubauer Improved) and a compound microscope. One sample was removed from each flask for counting. One or more hemacytometer fields, each 0.10 x 0.10 cm in surface area and 0.010-cm deep and containing 0.00010 mL of culture, were examined for each sample until at least 400 algal cells or four fields were counted. Observations of the health of the algal cells were made at each 24-hour interval.

- Monitoring of test conditions: Temperature was measured continuously with a VWR minimum/maximum thermometer located in a flask of water
adjacent to the test flasks in the environmental chamber. Minimum and maximum temperatures and the shaking rate of the orbital shaker were recorded daily. Light intensity was measured at four locations around the perimeter of the shaker table with a VWR Traceable light meter at 0 hour and at each 24-hour interval during the exposure period. Light intensity was measured in footcandles and converted to lux based on 1 footcandle = 10.76 lux. Photosynthetically-active radiation (PAR) of the test area was measured at test initiation using a Licor Model LI-189 photometer with sensor probe LI-190SA.

- Water quality: Water quality parameters (pH and conductivity) were measured at test initiation and at the termination of the 72-hour exposure period. Measurements at test initiation were conducted on the test solution prior to addition to the replicate flasks. At test termination, after cell counts were completed, the three replicate solutions (A through C) for the treatment and control and the six solvent controls (A through F) were respectively composited for pH and conductivity measurements.

- Range-finding test: A preliminary range-finding exposure was conducted at Springborn Smithers at nominal trimethoxyphenylsilane concentrations of 0.010, 0.10 and 1.0 mg a.i./L, a control and a solvent (DMF) control. Two exposure vessels were established for each concentration and the controls. At test termination, cells exposed to all treatment levels tested and the control were observed to be normal. Following 72 hours of exposure, cell densities in the control and solvent control averaged 141 and 154 x 10E4 cells/mL, respectively. Cell densities in the 0.010, 0.10 and
1.0 mg a.i./L treatment levels averaged 109, 83 and 83 x 10E4 cells/mL, respectively.
Reference substance (positive control):
no
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 0.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
but exposure is to hydrolysis products
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 0.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
but exposure is to hydrolysis products
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 0.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 0.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: growth rate, biomass and yield
Details on results:
- Exponential growth in the control (for algal test): yes
Reported statistics and error estimates:
The EC50 values (the concentration of test substance which reduced total yield, total biomass and average growth rate by 50% relative to the solvent control) were determined for the 72-hour observation interval for total biomass, average growth rate and total yield. If a sufficient response was observed, the EC values and their 95% confidence intervals were determined by linear regression of response (percent reduction of total biomass, average growth rate and total yield as compared with the solvent control) versus the initial concentration. A computer program, TOXSTAT® (Gulley et al., 1996), was used to assist in these computations. If less than the designated percent inhibition was observed for the noted parameter, the EC value was empirically estimated to be greater than the highest concentration tested.

The No-Observed-Effect Concentration (NOEC), the highest test concentration which demonstrated no statistically adverse effect (p ≤ 0.05) for each parameter when compared to the solvent control data, was determined. If the data sets passed the test for homogeneity and normality, Bonferroni’s t-Test was used to determine the NOEC. If the data did not pass the tests for homogeneity and normality, then Kruskal-Wallis’ Test 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.

Table 1. Results of analysis of test media

 

Nominal concentration (mg/L)

Measured concentration at 0 hours (mg/L)

Measured concentration at 0 hours as percentage of nominal

Measured concentration at 24 hours (mg/L)

Measured concentration at 24 hours as percentage of nominal

0 (Control)

<0.00017

Not applicable

<0.00018

Not applicable

0 (Solvent control)

<0.00017

Not applicable

<0.00018

Not applicable

0.013

0.012

90

<0.00018

Not applicable

0.025

0.016

64

<0.00018

Not applicable

0.050

0.033/0.045*

67/89

<0.0018/<0.0018

Not applicable

0.10

0.084

84

<0.0018

Not applicable

0.20

0.17

87

<0.0018

Not applicable

*Result of the additional sample without algae present to determine biological uptake/degradation

 

Table 2. Test results

 

Nominal concentration (mg/L)

Mean growth rate 0-72 h (/day)

Inhibition of growth rate (%)*

Mean total biomass as area under the growth curve ( 10E4 cell days/mL)

Inhibition of biomass (%)*

Mean yield (10E4 cells/mL)

Inhibition of yield (%)*

0 (Control)

1.69

-

96.28

-

129.50

-

0 (Solvent control)

1.72

-

100.54

-

141.22

-

0.013

1.72

0

103.60

-3

139.83

1

0.025

1.74

-1

104.49

-4

147.22

-4

0.050

1.72

0

102.37

-2

141.00

0

0.10

1.73

-1

101.86

-1

142.11

-1

0.20

1.72

0

103.89

-3

139.11

1

*Relative to solvent control. A negative value indicates that the value of the parameter was higher than for the solvent control.

Validity criteria fulfilled:
yes
Conclusions:
A 72 hour EC50 value of >0.2 mg/L and NOEC of ≥0.2 mg/L have been determined for the effects of the test substance on growth rate of Pseudokirchnerella subcapitata. The same results were obtained for biomass and yield endpoints. Analysis of the test media showed that measured concentrations of the parent substance declined to below the Limit of Quantification (LOQ) of the analytical method in all treatments within 24 hours of the start of the test. The substance is subject to rapid hydrolysis and it is therefore likely that the test organisms were primarily exposed to hydrolysis products retained in the test media.

Description of key information

QSAR (96-h) EC50 of 120 mg/l derived for the hydrolysis product, phenylsilanetriol

Key value for chemical safety assessment

Additional information

A 72 hour EC50value of >0.2 mg/l and NOEC of ≥0.2 mg/l have been determined for the effects of the test substance on growth rate of Pseudokirchnerella subcapitata (OECD Guideline 201 Alga, Growth Inhibition Test, Springborn Smithers 2008). The same results were obtained for biomass and yield endpoints. Analysis of the test media showed that measured concentrations of the parent substance declined to below the Limit of Quantification (LOQ) of the analytical method in all treatments within 24 hours of the start of the test. As the test was conducted well below the limit of solubility of the substance, the effect concentrations do not give an accurate representation of the toxicity. Therefore, it was deemed suitable to read across from the structurally analogous substance, trichloro(phenyl)silane (98 -13 -5) as both substances hydrolyse rapidly to form phenylsilanetriol. No effects for either growth rate or biomass were observed at the highest test concentration of 100 mg/l (OECD Guideline 201 Alga, Growth Inhibition Test, Springborn Smithers, 2009). The substance is subject to rapid hydrolysis and it is therefore likely that the test organisms were primarily exposed to hydrolysis products retained in the test media.

An EC50of 120 mg/l was derived for the hydrolysis product using an appropriate calculation method.