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Short-term toxicity to aquatic invertebrates

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Endpoint:
short-term toxicity to aquatic invertebrates
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:
48 h
Dose descriptor:
EC50
Effect conc.:
600 mg/L
Conclusions:
A 48 h LC50 value of 600 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:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2009-10-28 to 2009-10-30
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
- Sampling method: At test initiation, samples were removed from the intermediate mixing vessels for analysis of the hydrolysation product (trihydroxy(phenyl)silane). At test termination, the replicate solutions from the treatment levels and the control were composited within the treatments prior to sampling.
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 304 μL of Trichloro(phenyl)silane with 400 μL of tetrahydrofuran (THF) using a Hamilton syringe. A 100 mg test item/L stock solution was prepared prior to test initiation by adding 352 μL of the dosing stock solution to 1600 mL dilution water using a Hamilton syringe. Prior to addition of the dosing stock solution, the glass beaker containing the dilution water was placed on a magnetic stirrer. The spiked solution was stirred continuously over night. The pH of the solution was then adjusted to 7.01 with 1 N hydrochloric acid (HCl) and 1 N sodium hydroxide (NaOH). Thereafter, the stock solution was further diluted to a final volume of 2000 mL with dilution water, resulting in a solvent (THF) concentration of 0.10 mL/L. Nominal concentrations of 2.0, 4.3, 9.4, 20.7, 45.5 and 100 mg/L were prepared by diltion 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):
Daphnia magna
Details on test organisms:
TEST ORGANISM

- Source: The Daphnia magna used in this toxicity test were obtained from laboratory cultures maintained at Springborn Smithers Laboratories (Europe), originally obtained from a culture at Springborn Smithers Laboratories Wareham (MA), USA.

- Culture conditions: Prior to testing, the daphnids were held in glass beakers under a photoperiod of 16 hours light and 8 hours darkness with a 30 minute transition period.

- Culture medium: The culture water consisted of modified Elendt M4 medium. After preparation the medium was aerated using an air pump to bring the pH and dissolved gases into equilibrium with the atmosphere. The modified Elendt M4 medium was allowed to equilibrate for at least two days prior to use. Total hardness, total alkalinity, pH and specific conductivity of the dilution water were determined after preparing each batch to verify that these parameters are within the acceptable ranges.

- Feeding: During culture the daphnids were fed 1.5 - 2.0 mL of a solution containing approximately 4 x 107 cells/mL of the unicellular green alga, Ankistrodesmus falcatus (ANK) and approximately four drops or 0.5 mL of a combination of yeast, cereal leaves and flaked fish food (YCT) daily.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
150 mg/L as CaCO3
Test temperature:
19.1 to 21.2°C
pH:
7.31 to 7.94
Dissolved oxygen:
8.00 to 8.61 mg/L
Salinity:
Not applicable
Nominal and measured concentrations:
Nominal test concentrations: 0 (Control), 0 (solvent control), 2.0, 4.3, 9.4, 20.7, 45.5 and 100 mg/L

The concentration of the test item (determined as trihydroxy(phenyl)silane) in the newly prepared solutions at test start ranged from 92.9 to 103% of nominal concentrations. The concentration of the test item in the aged solutions at test termination (hour 48) ranged from 92.9 to 104% of nominal concentrations.

Based on these results, the nominal test concentrations were used for the evaluation of the biological data.
Details on test conditions:
TEST SYSTEM

- Replication: Four test vessels per treatment level and the controls were prepared.

- Age of test organisms: Daphnids, < 24 hours old

- Distribution of test organisms to treatments: Daphnids were impartially selected and distributed using an intermediate vessel by adding no more than two daphnids to each vessel until all intermediate vessels contained two daphnids. This procedure was repeated until each vessel contained 20 daphnids. The test was initiated when 5 daphnids were introduced to each replicate exposure vessel (replicates A to D). A total of 20 organisms were exposed to each treatment level and control solutions. Each test vessel was labeled with the study number, concentration and replicate designation.

- Feeding: The daphnids were not fed during the 48 hour exposure period.

- Dilution water: The batch of modified Elendt M4 medium used for the definitive test had a total hardness and alkalinity of 150 and 34 mg/L as CaCO3, respectively, a pH of 8.05 and a specific conductivity of 400 μS/cm.

- Observations: The number of immobilized daphnids observed in each replicate test vessel was recorded at test initiation and after 24 and 48 hours of exposure. Immobilization was defined as those animals not able to swim within 15 seconds after gentle agitation of the test vessel or by pipette. Biological observations (e.g., abnormal behavior or appearance of the test organisms) and observations of the physical characteristics of the test solutions (e.g., precipitate, film on the surface of the test solution) were also made and recorded after 0, 24 and 48 hours of exposure.

- Water quality: The pH, dissolved oxygen (DO) concentration and temperature were measured at 0, 24 and 48 hours in one replicate of each treatment level and the control. Continuous temperature monitoring was performed in an additional vessel adjacent to the test vessels throughout the exposure period. The pH was measured with a WTW 323 pH meter, dissolved oxygen and daily temperature were measured using a WTW Oxi 325 dissolved oxygen meter. Light intensity was measured with a Pancontrol LX 1308 lux meter.
Reference substance (positive control):
no
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
9.4 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
- Immobilisation of Control: 0 in Control, 5% in solvent Control
Reported statistics and error estimates:
There were insufficient levels of immobilisation in the treatments to derive an EC50 value.

The No-Observed-Effect Concentration (NOEC) was determined by visual observation of the raw data and is defined as the highest concentration tested at which there was no toxicant-related immobilization or physical and behavioral abnormalities (e.g., lethargy) with respect to the control organisms.

Table 1. Results of analysis of test media (measured as Trihydroxy(phenyl)silane)

 Nominal test substance concentration (mg/L)  Initial measured concentration (mg/L) and (Percentage of nominal) Measured concentration after 48 hours (mg/L) and (Percentage of nominal) 
 0 (Control)  <LOQ  <LOQ
 0 (Solvent control)  <LOQ  <LOQ
 2.0  1.86 (92.9)  1.86 (92.9)
 4.3  4.22 (98.1)  4.18 (97.1)
 9.4  9.22 (98.1)  9.49 (101)
 20.7  20.5 (99.0)  20.5 (99.2)
 45.5  46.8 (103)  47.5 (104)
 100  99.0 (99.0)  97.6 (97.6)

Table 2. Test results

 Nominal test substance concentration (mg/L)  Mean percentage immobilisation after 24 hours  Mean percentage immobilisation after 48 hours
 0 (Control)  0  0
 0 (Solvent control)  0  5
 2.0  0  0
 4.3  0  5
 9.4  0  0
 20.7  0  15
 45.5  0  15
 100  0  25
Validity criteria fulfilled:
yes
Conclusions:
A 48-hour EC50 value of >100 mg/L and a NOEC of 9.4 mg/L have been determined for the effects of the test substance on mobility of Daphnia magna. It is likely that the test organisms were predominantly exposed to the hydrolysis products of the test substance.
Endpoint:
short-term toxicity to aquatic invertebrates
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
GLP compliance:
yes (incl. certificate)
Salinity:
Not applicable
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2008-05-28 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 202 (Daphnia sp. Acute Immobilisation Test)
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
- Nominal concentrations: 0 (Control), 0 (Solvent control) and 0.20 mg a.i./L.

- Sampling method: Prior to the start of the definitive exposure, water samples were removed from the treatment level and the controls and analyzed for trimethoxyphenylsilane concentration. Results of these pretest analyses were used to judge whether sufficient quantities of trimethoxyphenylsilane were being delivered to the test vessels and whether the appropriate test concentrations were being maintained in order to initiate the definitive exposure.

During the in-life phase of the definitive study, water samples were collected from the treatment vessels and the controls and analyzed for trimethoxyphenylsilane at 0 hour (test initiation) and 48 hours (test termination). Samples used for analysis of trimethoxyphenylsilane were taken from alternating replicates (i.e., replicates C and D at test initiation and replicates A and B at test termination). Samples were collected from the approximate midpoint of the test vessel by pipette.

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.
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION

- Method: A 2.0 mg a.i./mL stock solution was prepared by placing 0.0508 g of trimethoxyphenylsilane in a 25-mL volumetric flask and bringing it to volume with DMF (CAS No. 68-12-2). The resulting stock solution was observed to be clear and colorless with no visible undissolved test substance.

A 0.51 mL/mL solvent stock solution was prepared by bringing 12.7 mL of DMF to a final volume of 25 mL with deionized water. The resulting stock solution was observed to be clear and colorless.

Prior to test initiation, a Glenco® 20-mL gas-tight syringe in conjunction with a Harvard Syringe Pump was calibrated to deliver 0.0394 mL/cycle of the 2.0 mg a.i./mL trimethoxyphenylsilane stock solution into the diluter system’s chemical mixing chamber which also received 0.394 L of dilution water per cycle. The mixing chamber was positioned over a water-driven magnetic stirrer which continuously mixed the contents of the chamber. The constant mixing aided in the solubilization of the test substance in the dilution water. The concentration of trimethoxyphenylsilane in the solution contained within the mixing chamber was equivalent to that of the nominal test concentration (0.20 mg a.i./L).

The solvent control solution was prepared utilizing a similar system, calibrated to deliver 0.0394 mL/cycle of the solvent control stock solution (0.51 mL/mL) to 0.20 L of dilution water per cycle which was subsequently delivered to the solvent control vessels. The concentration of DMF in the solvent control vessels was equivalent to the concentration of solvent present in the treatment level solution (0.10 mL/L).

The diluter system was calibrated prior to test initiation by measuring delivery volumes of test substance and dilution water. The function of the diluter system (e.g., flow rates, stock solution consumption) was monitored daily and a visual check of the system’s function was performed twice daily. In addition, analysis of the exposure solutions for trimethoxyphenylsilane concentration was used to verify proper operation of the diluter system. The exposure system was in proper operation for five days prior to test initiation to allow equilibration of the test substance in the diluter apparatus and exposure vessels.

Four glass tubes with an approximate length of 5 centimeters (cm) and an inside diameter of 2 millimeter (mm) were inserted through silicone stoppers in the flow-splitting chambers, entering individual glass delivery tubes. Delivery tubes were positioned to deliver solution to each replicate. At the end of each delivery tube, 1 mm (I.D.) glass capillary tubes were attached. These glass capillary tubes served to restrict the flow of the test solution and minimized potentially stressful turbulence in the exposure vessels. A total of 50 mL (i.e., one delivery tube per vessel) of test solution was delivered per cycle.
Test organisms (species):
Daphnia magna
Details on test organisms:
- Source: The Daphnia magna used in this toxicity test were obtained from laboratory cultures maintained at Springborn Smithers.

- Age of test organisms; Juvenile daphnids (< 24 hours old) were obtained from the laboratory cultures by removing all immature daphnids from the culture vessels (1.0-L glass beakers) 24 hours prior to test initiation, thereby isolating all mature gravid adult daphnids in the culture. Daphnids produced by these adult organisms were removed from the cultures and used as test organisms.

- Culture medium: The culture water was prepared by fortifying well water based on the formula for hard water (U.S. EPA, 1975) and filtering it through an Amberlite XAD-7 resin column to remove any potential organic contaminants. This water had a total hardness and total alkalinity range as calcium carbonate (CaCO3) of 170 mg/L and 100 to 110 mg/L, respectively, a pH range of 7.8 to 8.1, a dissolved oxygen concentration range of 7.5 to 9.0 mg/L, a temperature range of 20 to 22°C and a specific conductance of 550 micromhos per centimeter (μmhos/cm). All water quality ranges presented here were measured during the two weeks prior to testing.

- Lighting: The daphnid culture area received a regulated photoperiod of 16 hours of light and 8 hours of darkness. Light intensity of 54 to 71 footcandles (580 to 760 lux) at the surface of the culture solutions was provided by fluorescent bulbs.

- Feeding: Daphnids were fed a unicellular green algae (Ankistrodesmus falcatus, 4 x 10E7 cells/mL) at a rate of 1.0 to 2.0 mL per vessel daily depending on the age of the adult organisms in the culture vessel and 0.5 mL of a combination of yeast, cereal leaves and flaked fish food (YCT) daily. Daphnids were not fed during the exposure.
Test type:
flow-through
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Hardness:
160 mg/L as CaCO3
Test temperature:
20-21°C
pH:
7.0 - 8.3
Dissolved oxygen:
6.1-8.6 mg/L (>60% ASV)
Nominal and measured concentrations:
Nominal concentrations: 0 (Control), 0 (Solvent control) and 0.20 mg/L.

The mean measured concentration was defined as 0.0029 mg a.i./L. Due to the rapid hydrolysis of this compound combined with the limited solubility, these recoveries are considered to be the maximum achievable recoveries under testing conditions.

The results are interpreted with reference to nominal and mean measured exposure concentrations.
Details on test conditions:
- Exposure system: The toxicity test was conducted using an exposure system consisting of an intermittent-flow proportional diluter (Mount and Brungs, 1967) and a set of 12 exposure vessels. The exposure system was designed to provide one concentration of the test substance, a solvent (dimethylformamide (DMF, CAS No. 68-12-2)) control and a dilution water control. Test vessels were impartially placed in a temperature controlled water bath designed to maintain the test solution at a temperature of 20 ± 1 ºC.

The diluter system was calibrated prior to test initiation by measuring delivery volumes of test substance and dilution water. The function of the diluter system (e.g., flow rates, stock solution consumption) was monitored daily and a visual check of the system’s function was performed twice daily. In addition, analysis of the exposure solutions for trimethoxyphenylsilane concentration was used to verify proper operation of the diluter system. The exposure system was in proper operation for five days prior to test initiation to allow equilibration of the test substance in the diluter apparatus and exposure vessels. Four glass tubes with an approximate length of 5 centimeters (cm) and an inside diameter of 2 millimeter (mm) were inserted through silicone stoppers in the flow-splitting chambers, entering individual glass delivery tubes. Delivery tubes were positioned to deliver solution to each replicate. At the end of each delivery tube, 1 mm (I.D.) glass capillary tubes were attached. These glass capillary tubes served to restrict the flow of the test solution and minimized potentially stressful turbulence in the exposure vessels. A total of 50 mL (i.e., one delivery tube per vessel) of test solution was delivered per cycle.

- Test vessels: Each test vessel (1600-mL square glass battery jars) had two 2-cm holes drilled in the sides, which were covered with Nitex® 40-mesh screen for drainage. The total test solution volume was maintained at 1400 mL to ensure at least 2 mL of test solution was provided for each daphnid as required by the OECD guideline (OECD, 2004). Each replicate vessel received an average of 6 solution volume replacements per day in order to provide a 90% test solution volume replacement rate of approximately 9 hours (Sprague, 1969). Exposure vessels were labeled to identify the nominal test substance concentration and designated replicate.

- Replication: Four replicate vessels were established for the treatment level and each control. Daphnids were added impartially to intermediate beakers by adding no more than two daphnids at a time to each vessel until all beakers contained two daphnids. This process was repeated until each of the intermediate beakers contained 10 daphnids. The test was initiated when 10 daphnids (< 24 hours old) were introduced to each replicate exposure vessel. A total of 40 organisms were exposed to the treatment level and each control solution.

- Dilution water: The dilution water that was used during the definitive test was from the same source as the water in the daphnid cultures and was characterized during the course of the exposure as having a total hardness and total alkalinity as CaCO3 of 160 mg/L and 100 mg/L, respectively, a specific conductivity of 600 μmhos/cm, a pH of 7.9, and a dissolved oxygen concentration of 9.6 mg/L. The TOC concentration of the dilution water was 0.52 mg/L for the month of May 2008.

- Lighting: A 16-hour light, 8-hour dark photoperiod was maintained. Light intensity of 54 to 71 footcandles (580 to 760 lux) at the surface of the culture solutions was provided by fluorescent bulbs.

- Observations: The number of immobilized daphnids observed in each replicate test vessel was recorded at test initiation, 24 and 48 hours of exposure. Daphnids were determined immobile if they were not able to swim within 15 seconds after gentle agitation of the test vessel. Biological observations (e.g., abnormal behavior or appearance of the test organisms) and observations of the physical characteristics of the test solutions (e.g., precipitate, film on the surface of the test solution) were also made and recorded at 0, 24 and 48 hours of exposure.

- Water quality: The pH, dissolved oxygen concentration and temperature were measured once daily in each replicate vessel of each treatment level and the control throughout the exposure period. Temperature was also continuously monitored in replicate C of the 0.20 mg a.i./L nominal treatment throughout this study.

- Range-finding test: An initial 48-hour preliminary range-finding exposure was initiated under flow-through conditions at nominal concentrations of 0.013, 0.025, 0.050, 0.10 and 0.20 mg a.i./L, a control and a solvent control (two replicates each, ten daphnids per replicate). Following 48 hours of exposure, immobilization of 20% was observed in the 0.025 mg a.i./L treatment level, immobilization of 10% was observed in the 0.050 and 0.10 mg a.i./L treatment levels. One daphnid in the 0.025 mg a.i./L treatment level was observed to be caught on the screen of the test vessel. No immobilization or adverse effects were observed in the 0.013 and 0.20 mg a.i./L treatment levels or the controls. Due to the lack of a dose response in the high test concentration and the functional solubility of the test substance, a single concentration of 0.20 mg a.i./L, which was slightly above the functional solubility of the test substance in well water, was selected for the definitive test
Reference substance (positive control):
no
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
>= 0.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
but exposure is to hydrolysis products
Basis for effect:
mobility
Duration:
48 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:
mobility
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
>= 0.003 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 0.003 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
- Control immobilisation: Following 48 hours of exposure, 13% immobilization was observed in the control, however since the immobilization was limited to replicate C of the control and no immobilization was observed in the remaining three replicates of the control, solvent control or the treatment level tested, this immobilization was not considered representative of this exposure and is attributed to contaminated glassware.
Reported statistics and error estimates:
Since this test was conducted as a limit test (one concentration) and the concentration tested did not produce ≥ 50% immobilization, the EC50 value was empirically estimated to be greater than 0.20 mg a.i./L, the nominal concentration tested.

The No-Observed-Effect Concentration (NOEC) during the 48-hour exposure period was also determined. The NOEC is empirically determined and is defined as the highest concentration tested at which there were no toxicant-related immobilization or physical and behavioral abnormalities (e.g., lethargy), with respect to the control organisms.

Table 1. Results of analysis of test media

 

Nominal concentration (mg/L)

Mean measured concentration (mg/L)

Mean measured concentration as percentage of nominal

0 (Control)

<0.00017*

-

0 (Solvent control)

<0.00017*

-

0.20

0.0029

1.4

*The limit of quantification (LOQ) of the analytical method was 0.00017 mg/L

 

Table 2. Test results

 

Nominal concentration (mg/L)

Mean percentage immobilisation after 24 hours

Mean percentage immobilisation after 48 hours

0 (Control)

5

13*

0 (Solvent control)

0

0

0.20

0

0

*All observed immobilization was isolated to replicate C and is likely due to contaminated glassware. Since no immobilization was observed in the remaining three replicates of the control, the solvent Control or the treatment level tested, this immobilization was not considered to have impacted the interpretation of the exposure.

Validity criteria fulfilled:
yes
Conclusions:
A 48 hour EC50 value of >0.20 mg/L and NOEC of ≥0.20 mg/L have been determined for the effects of the test substance on mobility of Daphnia magna. The results are expressed relative to nominal test substance concentration. The corresponding mean measured concentration of the substance in the treated test medium over the course of the test was 0.0029 mg/L. However 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 600 mg/l derived for the hydrolysis product, phenylsilanetriol

Key value for chemical safety assessment

Additional information

A 48 hour EC50value of >0.20 mg/l and NOEC of ≥0.20 mg/l have been determined for the effects of the test substance on mobility of Daphnia magna (OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test, Springborn Smithers 2008). The results are expressed relative to nominal test substance concentration. The corresponding mean measured concentration of the substance in the treated test medium over the course of the test was 0.0029 mg/l. As the maximum concentration used in the study (0.2 mg/l) is lower than the solubility of the substance (1700 mg/l) the study does not provide an accurate representation of the toxicity of the substance. Therefore, it was considered appropriate to read across from the structurally analogous substance, trichloro(phenyl) silane (98-13-5) as both substances hydrolyse rapidly to phenylsilanetriol. No effects were observed to Daphnia magna at the highest concentration tested of 100 mg/l (OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test, Springborn Smithers 2009).

As the substance is subject to rapid hydrolysis, it is therefore likely that the test organisms were primarily exposed to hydrolysis products retained in the test media.

An EC50of 600 mg/l was derived for the hydrolysis product, based on an appropriate calculation method.