tert-butyl 3,5,5-trimethylperoxyhexanoate

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

disregarded due to major methodological deficiencies

Study period:

2003-06-16 to 2003-06-26

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Due to borderline effects observed and questionable test conditions taken the instability of the test item into account, the validity of this first algal growth inhibition study with tert-butyl 3,5,5-tris(methylperoxy)hexanoate was called into question (disregarded study). The reliability is categorised as Klimisch 4 (not assignable), as a final evaluation and EC50 value deduction is not possible. Thus, a further study was initiated to clarify the effects especially at the trigger value of 1 mg/L (key study).

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

1984

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Vehicle:

no

Details on test solutions:

A stock solution of the test item diluted in water was prepared at three days the beginning of the test (time 0) by mixing 100 mg of TERTIOBUTYL 3,5,5-TRIMETHYLPEROXYHEXANOATE in one litre of dilution water at 20°C.
Since the test was performed using hermetically closed flask, NaHCO3 0.3 g/L and HEPES buffer 6 mM (4-(2-Hydroxyethyl)-1 piperazineethanesulfonic acid, sodium salt) were added to dilution water.
Preliminary test: Volumetric flasks were filled with the required volumes of the test item stock solution and dilution water in order to obtain the test solutions at concentrations varying from 0.1 mg/L to 100 mg/L.
Definitive test: Volumetric flasks were filled with adequate volumes of the test item stock solution and dilution water in order to obtain the test solutions at concentrations varying from 0.5 mg/L to 20 mg/L. The definitive test was performed to a concentration range forming a geometric progression with a factor of 2.1

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata, CCAP 278/4 stock
- Source (laboratory, culture collection): Culture Centre of Algae and Protozoa (Ambleside, UK)

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

23 ± 1°C

pH:

7.57 - 8.49

Dissolved oxygen:

8.2 - 13.8 mg/L

Nominal and measured concentrations:

Nominal: 0.5, 1, 2.2, 4.5, 9.5, 20 mg/L
Initial (measured in non inoculated solutions): 0.05, 0.12, 0.26, 0.62, 1.8, 3.3 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: The study was performed using 120 ml glass bottles stoppered with PTFE bungs and sealed with aluminium caps
- Density of pre-culture: preliminary test: about 8.46 E6 cells/ml; definitive test: 3.15 E6; the cell density was measured daily
- The appearance of the test solutions was visually checked at the beginning and at the end of the test.
- Dissolved oxygen and pH were measured in the test solutions at the beginning and at the end of the test.
- Analytical chemistry and physico-chemical measurements were carried out at the beginning and the end of the test

TEST MEDIUM / WATER PARAMETERS
- Water: ultra-pure water used for the preparation of dilution water (resistivity > 18MΩ)

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.14 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% CL: 0.068 - 0.22

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.045 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% CL: 0.013 - 0.085

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.04 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.33 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 0.25 - 0.45

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 0.066 - 0.14

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.11 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

The appearance of the test solutions was visually checked at the beginning and at the and of the test. Solutions were found to be clear, colourless over the period of the test. No precipitation was observed at the end of the test.
Microscopic observation confirmed that the algae is a crescent chapped cell with an average length of 5-10µm.
During the test, the control pH varied by 1 unit.
The increase in cell density (R), measured in the control solution between the end and the beginning of the test, was greater than a factor of 16 (R=208).
The final concentrations of TERTIOBUTYL 3,5,5 -TRIMETHYLPEROXYHEXANOATE were maintained within the designated limit of 80 % of the initial concentrations in test flasks.

Results with reference substance (positive control):

The R value of the control was 280 which is higher than the minimal value mentioned by the quality criteria.

Reported statistics and error estimates:

Bartlett's test p-value for equality of variances= 0.091

Dissolved oxygen and pH were measured in the test solutions at the beginning and the end of the test:

Loading rate (mg/L)	pH*		Dissolved O2 (mg/L)
	T0	T72h	T0	T72h
0	7.57	7.68	8.2	8.6
0.04	7.61	8.42	8.6	13.8
0.11	7.61	8.3	8.6	13.2
0.16	7.61	7.95	8.6	11.4
0.31	7.59	7.75	8.6	8.9
0.94	7.59	7.75	8.6	8.7
1.59	7.57	7.64	8.6	8.7
T	7.57	8.49	8.5	13.7

*Measurements were carried out in non inoculated solutions at T0 and in inoculated solutions after 72 hours of incubation

The average percentage inhibition of cell growth (IAi) and growth rate (Iµi) are presented in the following table:

Loading rate (mg/L)	IAi (%)	Iµi (%)
0	0	0
1.5	98.95	92.4
0.94	98.73	89.69
0.31	94.96	62.42
0.16	62.29	23.43
0.11	26.05	7.69
0.04	5.79	1.34

Validity criteria fulfilled:

yes

Conclusions:

In this 72 -h algal growth inhibition test the EC50 (72 h) based on growth rate was determined to be 0.33 mg/L. The 72-h EC50 based on yield was 0.14 mg/L. The overall NOEC was determined to be 0.04 mg/L.

Executive summary:

Tert-Butylperoxy-3,5,5-trimethylhexanoat was tested in aquatic toxicity test to green algae Pseudokirchneriella subcapitata according to EU-Method C.3 and OECD guideline 201 (1984).

The concentration of test item which results in a 50% reduction in either cell growth (EbC50) or growth rate (ErC50) of the culture relative to the control is, if possible, estimated over a period of time of 72 hours. The no observed effect concentration (NOEC), highest tested concentration at which no significant inhibition of growth is observed relative to the control, is also estimated.

Algae were exposed to a range of concentrations of tert-Butylperoxy-3,5,5-trimethylhexanoat dissolved in dilution water. The toxic effect measured during the assay was the inhibition of cellular multiplication over a time period of 72 hours.

The study was performed using 120 ml glass bottles stoppered with PTFE bungs and sealed with aluminium caps, containing 50 ml of test solution inoculated with an algal suspension so that the initial cell concentration was equal to 1x 10⁴cells/mL. Test flasks were incubated at 23 ± 0.5°C continuously shaken and constantly illuminated. The cell density was measured daily. Analytical chemistry and physico-chemical measurements were carried out at the beginning and the end of the test. The following nominal test solution concentrations were used in the definitive test: 0.5, 1, 2.2, 4.5, 9.5, 20 mg/l. Effective concentrations and NOEC have been calculated using the geometric mean concentrations of tert-Butylperoxy-3,5,5-trimethylhexanoat. Concentrations of tert-Butylperoxy-3,5,5-trimethylhexanoat were measured by gas chromatography. Initial and final concentrations weren't equivalent to nominal concentrations.

The results were as follows:

Effective concentration and NOEC (mg/L)
	Value	95%CI
Cell growth
EbC50-72h	0.14	0.068 – 0.22
EbC10-72h	0.045	0.013 – 0.085
NOECb	0.04
Growth rate
ErC50-72h	0.33	0.25 – 0.45
ErC10-72h	0.1	0.066 – 0.14
NOECr	0.11

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011-01-10 to 2011-01-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

Commission Regulation (EC) No 761/2009

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

adopted 23rd March, 2006

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: At the start of the experiment three parallel samples were taken from each concentration level and from the control. At the end one sample was taken from each replicate test vessel and one sample was taken from three control container. Representative samples (in duplicate series) were taken at the start and at the end of the experiment.
- Sample storage conditions before analysis: One series was stored frozen (~ -20°C) in the testing laboratory, one series was sent for analysis and was analysed immediately after sampling.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test item stock solution was prepared as following: 25.16 mg test item was weighted and suspended in 500 mL dilution water (Mineral Salts Test Medium), thereafter stirred for 2 hours using a magnetic stirrer. After the 2-hour stirring the resulted “stock solution” was centrifuged at 4000 rpm for 10 min. (20°C) and filtrated using a 1.2 μm filter (Whatman® Glass Microfiber Filter) and further diluted accordingly.
- Eluate: water (mineral salts test medium)
- Controls: yes (untreated control: algal mineral salts test medium; toxic reference item: For the evaluation of the quality of the algae and the experimental conditions potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions).

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Source: Collection of Algal Cultures, Inst. Plant Physiology, University of Göttingen, Nikolausberger Weg 18, D-37073 Göttingen, GERMANY
- Method of cultivation: The stock cultures are small algal cultures that are planted on agar regularly. These are transferred to fresh medium at least once every two months under standardised conditions according to the test guidelines.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

22.5 - 23.2 °C

pH:

pH 7.36 to 7.90 at test start; pH 7.91 to 8.82 at test end

Nominal and measured concentrations:

- geometric mean of the measured concentrations: 0.125, 0.250, 0.516, 1.049, 2.029 mg/L
- nominal concentration: TISS/64, TISS/32, TISS/16, TISS/8, TISS/4 mg/L (TISS; Test item stock solution)

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks of 250 mL volume with 100 mL test medium
- Initial cells density: 10000 algal cells per mL test medium
- Control end cells density: 595000/mL
- No. of organisms per vessel: 100 mL algal suspension per replicates
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The algal mineral salts test medium was prepared according to OECD guideline No. 201
- Culture medium different from test medium: no
- Intervals of water quality measurement: Temperature: The cultures were maintained at a temperature in the range of 21 – 24 °C controlled at ± 2 °C, which was checked at the beginning of the study and every 24 hours in a flask filled with water. In addition, the temperature was continuously measured (with a min/max thermometer) within the climate chamber.; pH: The pH was checked at the beginning and at the end of the study in the controls and the treated variants.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: yes
- Light intensity and quality: approx. 8000 lux (in average of 8049 lux)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: The cell number was determined at 24, 48 and 72 hours after starting the test by manual cell counting using a microscope with a counting chamber.

TEST CONCENTRATIONS
- Range finding study: The concentration levels are chosen based on the results of performed range-finding tests.
- Test concentrations: In the first preliminary range-finding test the nominal concentration range of: 0.39; 0.78; 1.56; 3.125; 6.25; 12.5; 25 and 50 mg/L concentrations were tested, however instead of the nominal concentrations a more precise description was applied: TISS/128; TISS/64; TISS/32; TISS/16; TISS/8; TISS/4; TISS/2 and TISS mg/L (TISS: Test Item Stock Solution). In the second preliminary range finding test the investigated (nominal) concentrations were: TISS/64; TISS/16; TISS/8 and TISS/4.
- Results used to determine the conditions for the definitive study: In the first preliminary range finding test (after 72 hours) an inhibition of 100 % at a concentration of TISS/4 was determined. In the second preliminary range finding test (after 72 hours) an inhibition of 85.1 % at a concentration of TISS/4 was determined.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.323 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 0.2895 – 0.3595 (95 % conf. limits)

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.51 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 0.4577 – 0.5679 (95 % conf. limits)

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.125 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.125 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.25 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.25 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

The shape of the algal cells growing was obviously not affected in the concentration range of 0.125-0.516 mg/L (TISS/64-TISS/16 mg/L) and fragmented cells were observed at the concentrations of 1.049 and 2.029 mg/L (TISS/8 and TISS/4 mg/L).
In the control the cell density has increased from nominal N= 1 x 10000 cells/mL at the start of the test (0 hours) to N= 59.50 x 10000 cells/mL (mean value) after 72 hours by a factor of 59.50 . Thus, the algal growth in the control was high enough to pass the validity criteria in this assay.

Results with reference substance (positive control):

The experimental phase of the last study (Study Number: 392.820.2266) with reference item was: 16-19 August 2010. The report date: 10 September 2010.
The ErC 50 of Potassium dichromate: 1.05 mg/L, (95 % confidence limits: 0.96 – 1.16 mg/L)
The EbC 50 of Potassium dichromate: 0.63 mg/L, (95 % confidence limits: 0.57 – 0.69 mg/L)
The EyC 50 of Potassium dichromate: 0.56 mg/L, (95 % confidence limits: 0.51 – 0.61 mg/L)
The growth rate, 72h value is in accordance with the accepted 1.10 ± 0.48 mg/L range for Pseudokirchneriella subcapitata.

Reported statistics and error estimates:

Mean values and standard deviations were calculated for each treatment at the start, and at the end of the test using Excel for Windows software. Percentage inhibition of area (A), yield (Y) and growth rate (r) were calculated using EXCEL for Windows software. The EC50 values of the test item and their confidence limits were calculated using Probit analysis. The analysis was done using the TOXSTAT statistical software program. For the determination of the LOEC and NOEC, the calculated mean biomass b (area under the growth curve), growth rates r and yield at the test concentrations were tested on significant differences to the control values by Bonferroni t-Test and Williams’test by TOXSTAT software. The data were checked for normality by Chi-Square Test, for homogeneity of variance by Levene’s Test.

Validity criteria fulfilled:

yes

Conclusions:

In this 72-h algal growth inhibition test on Pseudokirchneriella subcapitata with the test item, the 72-h EC50 based on biomass was determined to be 0.3226 mg/L, based on the growth rate 0.5098 mg/L and based on the yield the 72-h EC50 was 0.2707 mg/L.
The overall NOEC was determined to be 0.125 mg/L (all calculations were based on the geometric mean of measured concentrations).

Executive summary:

Tert-Butylperoxy-3,5,5-trimethylhexanoat was tested in aquatic algae Pseudokirchneriella subcapitata according to EU-Method C.3 (2009) and OECD guideline 201 (2006).

At the start of the test the following test item concentrations were measured: TISS/64: 0.192 mg/L; TISS/32: 0.392 mg/L; TISS/16: 0.761 mg/L; TISS/8: 1.55 mg/L; TISS/4: 3.12 mg/L.

At the end of the test the measured concentrations were in the range of 41-46 % of the measured start concentrations. The following tert-Butylperoxy-3,5,5 -trimethylhexanoat concentrations were measured at the end of the test: TISS/64: 0.081 mg/L; TISS/32: 0.160 mg/L; TISS/16: 0.350 mg/L; TISS/8: 0.71 mg/L; TISS/4: 1.32 mg/L. The test item concentrations of the test solutions were measured by reverse phase HPLC method with UV detection at the start and at the end of the test. The test item had a statistically significant inhibitory effect on the growth based on the average specific growth rate, yield and areas under the growth curves of Pseudokirchneriella subcapitata after the exposure period of 72 hours in the concentration range of 0.250–2.029 mg test item/L (Bonferroni t-Test (and William’s test), α=0.05).The test concentration of 0.125 mg/L was determined as the 72-hour NOEC.

In the 72-h algal growth inhibition test on Pseudokirchneriella subcapitata with tert-Butylperoxy-3,5,5-trimethylhexanoat, the 72 -h EC50 based on biomass was determined to be 0.3226 mg/L, based on the growth rate 0.5098 mg/L and based on the yield the 72-h EC50 was 0.2707 mg/L.

The overall NOEC was determined to be 0.125 mg/L (all calculations were based on the geometric mean of measured concentrations).

Description of key information

Due to borderline effects observed and questionable test conditions taken the instability of the test item into account, the validity of the first algal growth inhibition study with tert-butyl 3,5,5-tris(methylperoxy)hexanoate was called into question (disregarded study). The reliability is categorised as Klimisch 4 (not assignable), as a final evaluation and EC50 value deduction is not possible. Thus, a further study was initiated to clarify the effects especially at the trigger value of 1 mg/L (key study).
In the disregarded study, the effect of the test item tert-Butylperoxy-3,5,5-trimethylhexanoat on the growth of fresh-water green algae Pseudokirchneriella subcapitata was assessed. The ErC50 (72 h) based on growth rate was determined to be 0.33 mg/L.
In the key study, a 72-h algal growth inhibition test on Pseudokirchneriella subcapitata with the test item, the 72-h EC50 based on biomass was determined to be 0.3226 mg/L, based on the growth rate 0.5098 mg/L and based on the yield the 72-h EC50 was 0.2707 mg/L.
The overall NOEC was determined to be 0.125 mg/L (all calculations were based on the geometric mean of measured concentrations). The results obtained in this second study were used for the risk assessment.

Key value for chemical safety assessment

EC50 for freshwater algae:

0.51 mg/L

EC10 or NOEC for freshwater algae:

0.125 mg/L

Additional information

Due to borderline effects observed and questionable test conditions taken the instability of the test item into account, the validity of the first algal growth inhibition study with tert-butyl-3,5,5-tris(methylperoxy)hexanoate was called into question (disregarded study). The reliability is categorised as Klimisch 4 (not assignable), as a final evaluation and EC50 value deduction is not possible. Thus, a further study was initiated to clarify the effects especially at the trigger value of 1 mg/L (key study). The results obtained in this second study were used for the risk assessment.

In the key study, tert-Butylperoxy-3,5,5-trimethylhexanoat was tested in aquatic algae Pseudokirchneriella subcapitata according to EU-Method C.3 (2009) and OECD guideline 201 (2006). At the start of the test the following test item concentrations were measured (TISS=Test Item Stock Solution): TISS/64: 0.192 mg/L; TISS/32: 0.392 mg/L; TISS/16: 0.761 mg/L; TISS/8: 1.55 mg/L; TISS/4: 3.12 mg/L.

At the end of the test the measured concentrations were in the range of 41-46 % of the measured start concentrations. The following tert-Butylperoxy-3,5,5-trimethylhexanoat concentrations were measured at the end of the test: TISS/64: 0.081 mg/L; TISS/32: 0.160 mg/L; TISS/16: 0.350 mg/L; TISS/8: 0.71 mg/L; TISS/4: 1.32 mg/L. The test item concentrations of the test solutions were measured by reverse phase HPLC method with UV detection at the start and at the end of the test. The test item had a statistically significant inhibitory effect on the growth based on the average specific growth rate, yield and areas under the growth curves of Pseudokirchneriella subcapitata after the exposure period of 72 hours in the concentration range of 0.250–2.029 mg test item/L (Bonferroni t-Test (and William’s test), α=0.05).The test concentration of 0.125 mg/L was determined as the 72-hour NOEC.

In the 72-h algal growth inhibition test on Pseudokirchneriella subcapitata with tert-Butylperoxy-3,5,5-trimethylhexanoat, the 72-h EC50 based on biomass was determined to be 0.3226 mg/L, based on the growth rate 0.5098 mg/L and based on the yield the 72-h EC50 was 0.2707 mg/L.

The overall NOEC was determined to be 0.125 mg/L (all calculations were based on the geometric mean of measured concentrations).

In the disregarded study, tert-Butylperoxy-3,5,5-trimethylhexanoat was tested in aquatic toxicity test to green algae Pseudokirchneriella subcapitata according to EU-Method C.3 and OECD guideline 201 (1984).

The toxic effect measured during the assay was the inhibition of cellular multiplication over a time period of 72 hours.

The study was performed using 120 ml glass bottles stoppered with PTFE bungs and sealed with aluminium caps, containing 50 ml of test solution inoculated with an algal suspension so that the initial cell concentration was equal to 1x10⁴cells/ml. Test flasks were incubated at 23 ± 0.5°C continuously shaken and constantly illuminated. The cell density was measured daily. Analytical chemistry and physico-chemical measurements were carried out at the beginning and the end of the test. The following nominal test solution concentrations were used in the definitive test: 0.5, 1, 2.2, 4.5, 9.5, 20 mg/l. Effective concentrations and NOEC have been calculated using the geometric mean concentrations of tert-butyl-3,5,5-trimethylperoxyhexanoate.

Concentrations of tert-Butylperoxy-3,5,5-trimethylhexanoat were measured by gas chromatography and the measured concentration were used for NOEC, LC50 calculations.

In this 72 -h algal growth inhibition test the EC50 (72 h) based on growth rate was determined to be 0.33 mg/L. The 72 -h EC50 based on yield was 0.14 mg/L The overall NOEC was determined to be 0.04 mg/L. All results were based on the nominal concentrations.