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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:
20 August - 9 November, 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
yes
Remarks:
1.) method validation (diluting the samples in 100% acetonitrile) 2.) Replicate D (flask containing no algae) was left in the same position throughout the duration of the test.
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 6.3, 13, 25, 50 and 100 mg/l
- Sampling method: At test initiation, 24 and 72 hours, a single sample was removed from each test solution and the control and analysed for the test substance. Samples analysed on day 0 were removed from the test solutions in the volumetric flasks prior to filling the individual test flasks. Samples analysed at 24 and 72 hours of exposure were removed from the composite of replicate vessels for each treatment and the control.

At 24 and 72 hours of exposure, a sample was removed from the replicate flask (D) of the 25 mg/L solution which did not contain algae. The result of this analysis was compared with the 24 and 72 hour analyses of the 25 mg/L solution containing algae to assess the impact that algae bad on the test substance concentration.

Three quality control (QC) samples each were prepared at each sampling interval and remained with the appropriate set of exposure solution samples throughout the analytical process. These QC samples were prepared in dilution water at nominal concentrations similar to the exposure concentration range. Results of the analyses of the QC samples were used to judge the precision and quality control maintained during the analysis of exposure solution samples.

- Sample storage conditions before analysis: Not reported
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Prior to test initiation, a 100 mg/L primary stock solution was prepared by placing 0.2066g (0.2000 g as active ingredient) of the test substance into a 2000-ml volumetric flask and bringing it to volume with AAP medium. The resulting stock solution was observed to be clear and colourless with a small amount of undissolved test substance. Following 10 minutes of mixing with a magnetic stir plate and Teflon® coated stir bar, and two minutes of sonication a small amount of undissolved material remained.
Exposure solutions were prepared using the 100mg/l primary stock solution as follows:
Primary stock concentration (mg/l Volume of Stock Used (ml) Diluted to (ml) with AAP medium Nominal concentration (mg/l)
100 300 NA 100
100 500 1000 50
100 250 1000 25
100 130 1000 13
100 63 1000 6.3

- Controls: Untreated AAP medium
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): n/a
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): n/a
- Evidence of undissolved material (e.g. precipitate, surface film, etc): Stock solution was clear and colourless with a small amount of undissolved test material. All test solutions, with the exception of the 100 mg/l test solution, were observed to be clear and colorless with no visible undissolved test substance after stirring with a glass rod. The 100 mg/L test solution was clear and colourless and still contained a smll amount of undissolved test material.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: 1648
- Source (laboratory, culture collection): University of Texas, Austin, Texas, and was maintained in stock culture at Smithers Viscient
- Age of inoculum (at test initiation): not reported
- Method of cultivation: Not reported

ACCLIMATION
- Acclimation period: The innoculum used to initiate the toxicity test with the test substance was taken from a stock culture that had been transferred to fresh medium three days before testing.
- Culturing media and conditions (same as test or not): Yes
- Any deformed or abnormal cells observed: Not reported
Test type:
static
Water media type:
other: deionized water with AAP medium
Limit test:
yes
Total exposure duration:
72 h
Test temperature:
24
pH:
6.8 - 8.0
Nominal and measured concentrations:
Nominal: Control, 6.3, 13, 25, 50 and 100 mgI
Initial Measured concentrations: control, 4.8, 9.7, 20, 40 and 90 mg/l
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Type (delete if not applicable): open / closed: All test vessels were fitted with stainless steel caps which permitted gas exchange.
- Material, size, headspace, fill volume: 250-mL Erlenmeyer flasks filled with One hundred milliliters
- Aeration: not reported
- Renewal rate of test solution (frequency/flow rate): Static
- Initial cells density: 1.0 x 10⁴ cells/mL
- Control end cells density:
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): n/a

GROWTH MEDIUM
- Standard medium used: yes (AAP)
- Detailed composition if non-standard medium was used: n/a

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Algal Assay Procedure (AAP) medium prepared with sterile deionised water.
- Total organic carbon: 0.66 mg/l
- Conductivity: 94-110 µS/cm
- Culture medium different from test medium: No
- Intervals of water quality measurement: pH and conductivity measured at test initiation and test termination. Light intensity was measured every 24 hours.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: yes
- Photoperiod: Continuous light
- Light intensity and quality: 4400 to 5900 lux (420 to 550 footcandles) and photosynthetically-active radiation (PAR) range of 60 to 120 µE/m2/s.


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : At each subsequent 24-hour interval, a single cell count was conducted on each replicate solution of the treatment levels 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 0.01 O-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.
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter]: hemacytometer (Neubauer Improved) and a compound microscope.


TEST CONCENTRATIONS
- Spacing factor for test concentrations: approx. 2.
- Justification for using less concentrations than requested by guideline: n/a
- Range finding study
- Test concentrations: 0.0010, 0.010, 0.10, 1.0 and 10 mg/L
- Results used to determine the conditions for the definitive study: Following 72 hours of exposure, cell densities in the 0.0010, 0.010, 0.10, 1.0 and 10 mg/L treatment levels averaged 61.00, 78.00, 61.50, 72.75 and 57.63 x 10⁴ cells/mL, respectively. The control and solvent control averaged 41.88 and 56.13 x 10⁴ cells/mL, respectively. Based on these results and consultation with the Study Sponsor, nominal concentrations of 6.3, 13, 25, 50 and 100 mg/L and a control were selected for the definitive exposure.
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
90 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 90 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Reported statistics and error estimates:
Bonferroni's adjusted t-test used to determine treatment related effects.

Additional testing to further define the EC50 values was not performed since the highest nominal concentration tested (100 mg/L) equals the maximum required test concentration in the study guideline.

The following acceptance criteria were required by the protocol: the cell growth in the control must increase from the initial density (1.0 x 104 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 control replicates should not exceed 35%. The CV for the average growth rate of the control for the entire test period (0- to 72-hour growth rate) should not exceed 7%. The results ofthis test are presented in the following table:

 Acceptance Criteria for Control  Study Result
 Exceed 16 x 10exp.4 cells/mL at 72 hours  111.58 x 10exp.4 cells/mL
 Mean daily growth rate CV < 35%  18%
 0 - 72 hour growth rate CV < 7%  3.9%

During this study, the 72-hour cell density in the control was 111.58 x 104 cells/rnL, which meets the above criterion. The mean daily CV for growth rates was 18% and the CV for 0- to 72-hour growth rate was 3.9%, which meet the above criteria.

Validity criteria fulfilled:
yes
Conclusions:
A 72-hour EC50 value of >90 mg/l and a NOEC value of 90 mg/l has been determined for the effects of 3-(trimethoxysilyl)propyl (2E,4E)-hexa-2,4-dienoate on growth rate of Pseudokirchneriella subcapitata, based on initial measured concentrations.
Executive summary:

The purpose of this study was to determine the effect of 2,4-Hexadienoic Acid, 3-(Trimethoxysilyl) Propyl Ester on the growth of the freshwater green alga Pseudokirchneriella subcapitata, during a 72-hour exposure. The test was designed to meet the testing requirements of the Organization for Economic Co-operation and Development (OECD) Guideline for the Testing of Chemicals #201, Freshwater Alga and Cyanobacteria, Growth Inhibition Test. The test was performed from August 20, 2012 to August 23, 2012. Pseudokirchneriella subcapitata was exposed to five test concentrations and a control. Nominal test concentrations were 6.3, 13, 25, 50 and 100 mg/L. Test concentrations were based on a preliminary range-finding exposure. Three replicates were tested per treatment group and the initial cell density was approximately 10,000 cells/mL. Algal Assay Procedure (AAP) medium was used to prepare the exposure solutions. At each subsequent 24-hour interval, a single cell count was conducted on each replicate solution of the treatment levels and the controls using a hemacytometer (Neubauer Improved) and a compound microscope. At test initiation, 24 and 72 hours, a single sample was removed from each test solution and the control and analysed for the test substance. All exposure solutions were analysed using a high performance liquid chromatographic system equipped with ultraviolet detection (HPLC/UV). The results are based on initial measured concentrations of the test substance. Environmental conditions during the study remained within acceptable limits. Continuous temperature monitoring established that the temperature was 24 degrees C during the test with continuous illumination at a range of 420 to 540 foot candles. The shaking rate was maintained at 100 rpm. Initial measured concentrations of the test substance ranged from 75 to 90% of nominal concentrations and defined the treatment levels tested as 4.8, 9.7, 20, 40 and 90 mg/L. At 24 hours of exposure, test solution concentrations ranged from 1.0 to 1.7% of nominal and at 72 hours, all test solutions were below the limit of quantitation. The test substance is known to hydrolyse rapidly in water and its stability is highly pH dependent so the decline in test substance concentration was expected. Algal cells exposed to all treatment levels and the control was observed to be normal throughout the exposure period. The 0- to 72-hour yield in the control averaged 110.58 x 104 cells/mL. The 0- to 72-hour yield in the 4.8, 9.7, 20, 40 and 90 mg/L treatment levels averaged 107.00, 120.58, 113.06, 120.25 and 100.25 x 104 cells/mL respectively. No significant reduction in yield was detected in all the treatment levels in comparison to the control data. The 0- to 72-hour growth rate in the control averaged 1.54 days-1. The 0- to 72-hour growth rate in the 4.8, 9.7, 20, 40 and 90 mg/L treatment levels averaged 1.53, 1.57, 1.54, 1.57 and 1.51 days-1, respectively. No significant reduction in growth rate was detected in all the treatment levels in comparison to the control data. Therefore, the 72-hour NOEC for yield and growth rate was 90 mg/L. Since no concentration tested resulted in > 50% reduction in yield or growth rate, the 72-hour EyC50 and ErC50 was empirically estimated to be >90 mg/L, the highest initial measured concentration tested.

Description of key information

Short-term toxicity to algae: 72-h EC50 >90 mg/l (highest concentration tested), NOEC 90 mg/l (initial measured concentration) (OECD 201). The EC50 is equivalent to >76 mg/l when expressed in terms of the hydrolysis product, 3-(trihydroxysilyl)propyl-(2E,4E)-2,4-hexadienoate.

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:
76 mg/L

Additional information

A 72 hour EC50 value of >90 mg/l (highest concentration tested) and a NOEC value of 90 mg/l (initial measured concentrations) have been determined for the effects of 3-(trimethoxysilyl)propyl-(2E,4E)-hexa-2,4-dienoate on growth rate of Pseudokirchneriella subcapitata.

In view of the static exposure regime it is likely that the test organisms were predominantly exposed to the hydrolysis products of the test substance.

The results may be expressed in terms of concentration of the hydrolysis product, 3-(trihydroxysilyl)propyl-(2E,4E)-2,4-hexadienoate, by applying a molecular weight correction: (MW of silanol = 232.31 / MW of parent = 274.37) * >90 = >76 mg/l as 3-(trihydroxysilyl)propyl-(2E,4E)-2,4-hexadienoate.

The measured concentration may be classed as a conservative result because analysis of exposure concentrations was specific to the parent substance. It may be appropriate to present the results in terms of the nominal concentration for assessment of the hydrolysis product:

The 72 h EC50 value for growth rate of Pseudokirchneriella subcapitata based on nominal concentrations, is >100 mg/l.

In terms of concentration of the hydrolysis product this is 232.31/274.37 * >100 = >85 mg/l as 3-(trihydroxysilyl)propyl-(2E,4E)-2,4-hexadienoate.

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