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Toxicity to aquatic plants other than algae

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Reference
Endpoint:
toxicity to aquatic plants other than algae
Type of information:
experimental study
Adequacy of study:
key study
Study period:
This study was conducted between 16 August and 04 October 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 221 (Lemna sp. Growth Inhibition Test)
Version / remarks:
March 2006
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Information as provided by the Sponsor.
Identification: FAT 65004/F TE
Physical state/Appearance: Beige solid
Batch: 15060201 (China)
Purity: 99.9%
Expiry Date: 25 August 2020
Storage Conditions: Room temperature in the dark
Analytical monitoring:
yes
Details on sampling:
Range-Finding Test
In order to determine the stability of the test item under test conditions a sample of each test concentration was taken for chemical analysis on Day 0 (fresh media) and Day 3 (old media). An additional sample of each test concentration was prepared on Day 0 and incubated alongside the test until Day 7 in order to determine stability over the entire test duration. All samples were stored frozen prior to analysis.

Definitive Test
Verification of Test Concentrations
Samples were taken from the control and 100% v/v saturated solution test group from the freshly prepared bulk test preparation on Day 0 and from the pooled replicates on Day 7 for quantitative analysis. All samples were stored frozen prior to analysis. Duplicate samples were taken at each occasion and stored frozen for further analysis if necessary.
Vehicle:
no
Details on test solutions:
Preliminary Media Preparation Trial
Preliminary solubility work conducted indicated that the test item was practically insoluble in water using traditional methods of preparation e.g. ultrasonication and high shear mixing.
Based on this information the test item was categorized as being a ‘difficult substance’ as defined by the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures (OECD 2000). Therefore a media preparation trial was conducted in order to determine the solubility of the test item under test conditions.

Saturated Solution Preparation
A nominal amount of test item (550 mg) was dispersed, in duplicate, in 11 liters of deionized reverse osmosis purified water with the aid of propeller stirring at approximately 1500 rpm for periods of either 24 or 48 hours. After stirring samples were taken for chemical analysis after the following pre-treatments:
• Centrifugation at 10000 g for 30 minutes
• Centrifugation at 40000 g for 30 minutes
• Filtration through a 0.2 μm Sartorius Sartopore filter (approximately 1 liter discarded in order to pre-condition the filter)
• Filtration through a 0.2 μm Sartorius Sartopore filter (approximately 2 liters discarded in order to pre-condition the filter)

Results
Stirring Period and Treatment Concentration Found (mg/L)
24 Hours Centrifuged 10000 g 0.255
24 Hours Centrifuged 40000 g 0.499
24 Hours Filtered ~ 1 liter discarded 24 Hours Filtered ~ 2 liters discarded 48 Hours Centrifuged 10000 g 0.314
48 Hours Centrifuged 40000 g 0.633
48 Hours Filtered ~ 1 liter discarded 48 Hours Filtered ~ 2 liters discarded
Discussion
It is evident from these results that filtration was not appropriate as measured concentrations of less than the Limit of Quantification (LOQ) of the analytical method suggest that the test item was adsorbing to the filter matrices.
The 48-Hour preparation gave the highest dissolved concentration after centrifugation at 40000 g (0.63 mg/L). Based on these results, for the purposes of the study, the test item was prepared as a saturated solution at an initial loading rate of 50 mg/L, stirred via propeller stirrer for 48 hours prior to the removal of any undissolved test item by centrifugation at 40000 g for 30 minutes to give a 100% v/v saturated solution with a nominal test concentration of approximately 0.63 mg/L.

Range-finding Test
The results obtained from the preliminary media preparation trial conducted indicated that a dissolved test item concentration of approximately 0.63 mg/L could be obtained using a saturated solution method of preparation.
The test concentration to be used in the initial experiment was determined by a preliminary range-finding test. The range-finding test was conducted by exposing Lemna minor to a series of nominal test concentrations of 1.0, 10 and 100% v/v saturated solution for a period of 7 days. The test was conducted in glass conical flasks (500 mL). Two replicate flasks each containing 250 mL were prepared for the control and each test concentration.
A nominal amount of test item (550 mg) was dispersed in 11 liters of culture medium with the aid of propeller stirring at approximately 1500 rpm for 48 hours. After 48 hours the stirring was stopped and any undissolved test item was removed by centrifugation at 40000 g to give a 100% v/v saturated solution. A series of dilutions was made from this saturated solution to give further test concentrations of 10 and 1.0% v/v saturated solution.
Each of the prepared concentrations was inverted several times to ensure adequate mixing and homogeneity.

Definitive Test
Based on the result of the range-finding test a “limit test” was conducted at a concentration of 100% v/v saturated solution to confirm that at the highest attainable concentration, no effect on growth was observed.

Experimental Preparation
A nominal amount of test item (550 mg) was dispersed in 11 liters of culture medium with the aid of propeller stirring at approximately 1500 rpm for 48 hours. After 48 hours the stirring was stopped and any undissolved test item was removed by centrifugation at 40000 g for 30 minutes to give a 100% v/v saturated solution.
The concentration and stability of the test item in the test preparations were verified by chemical analysis on Day 0 (fresh media) and on Day 7 (old media)



Test organisms (species):
Lemna minor
Details on test organisms:
The test was carried out using Lemna minor. A culture of Lemna minor was obtained from Canadian Phycological Culture Centre, University of Waterloo, Ontario, Canada. Cultures were maintained in the laboratory by the periodic replenishment of culture medium. The culture was maintained in the laboratory at a temperature of 24 ± 1 ºC under continuous illumination (intensity approximately 7000 lux) for at least 7 days prior to the start of the test.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
7 d
Test temperature:
24 ± 1 ”C
pH:
6.5 ± 2 ”C
Nominal and measured concentrations:
Verification of Test Concentrations
Chemical analysis of the freshly prepared 100% v/v saturated solution test preparation on Day 0 showed a measured test concentration of 0.18 mg/L was obtained. Analysis of the 100% v/v saturated solution test preparation on Day 7 showed a decline in measured test concentration to less than the limit of quantification (LOQ) of the analytical method employed which was determined to be 0.095 mg/L.
Given this decline in measured test concentration it was considered justifiable to base the results on the geometric mean measured test concentration in order to give a “worst case” analysis of the data. The geometric mean measured test concentration was determined to be 0.092 mg/L
Details on test conditions:
Range-finding Test
The results obtained from the preliminary media preparation trial conducted indicated that a dissolved test item concentration of approximately 0.63 mg/L could be obtained using a saturated solution method of preparation.
The test concentration to be used in the initial experiment was determined by a preliminary range-finding test. The range-finding test was conducted by exposing Lemna minor to a series of nominal test concentrations of 1.0, 10 and 100% v/v saturated solution for a period of 7 days. The test was conducted in glass conical flasks (500 mL). Two replicate flasks each containing 250 mL were prepared for the control and each test concentration.
A nominal amount of test item (550 mg) was dispersed in 11 liters of culture medium with the aid of propeller stirring at approximately 1500 rpm for 48 hours. After 48 hours the stirring was stopped and any undissolved test item was removed by centrifugation at 40000 g to give a 100% v/v saturated solution. A series of dilutions was made from this saturated solution to give further test concentrations of 10 and 1.0% v/v saturated solution.
Each of the prepared concentrations was inverted several times to ensure adequate mixing and homogeneity.
The control group was maintained under identical conditions but not exposed to the test item.
At the start of the range-finding test the number of fronds present in each test and control culture was recorded along with observations on frond size, appearance, root length and number of colonies present. The flasks were then incubated at 24 ± 1 ºC under continuous illumination (intensity approximately 7000 lux) for 7 days.
On Days 3 and 5 the test solutions were renewed, and observations on the test organisms were recorded on days 0, 3, 5 and 7.
In order to determine the stability of the test item under test conditions a sample of each test concentration was taken for chemical analysis on Day 0 (fresh media) and Day 3 (old media). An additional sample of each test concentration was prepared on Day 0 and incubated alongside the test until Day 7 in order to determine stability over the entire test duration. All samples were stored frozen prior to analysis.

Definitive Test
Based on the result of the range-finding test a “limit test” was conducted at a concentration of 100% v/v saturated solution to confirm that at the highest attainable concentration, no effect on growth was observed.
Experimental Preparation
A nominal amount of test item (550 mg) was dispersed in 11 liters of culture medium with the aid of propeller stirring at approximately 1500 rpm for 48 hours. After 48 hours the stirring was stopped and any undissolved test item was removed by centrifugation at 40000 g for 30 minutes to give a 100% v/v saturated solution.
The concentration and stability of the test item in the test preparations were verified by chemical analysis on Day 0 (fresh media) and on Day 7 (old media) (see Annex 4).
Exposure Conditions
As in the range-finding test glass conical flasks were used. Six flasks each containing 250 mL of solution were prepared for the control and 100% v/v saturated solution treatment group.
The control group was maintained under identical conditions but not exposed to the test item.
Each control and test flask was inoculated with 3 colonies of Lemna minor (total 12 fronds). The flasks were then incubated at 24 ± 1 ºC under constant illumination (intensity approximately 7000 lux) for 7 days.
A static testing regime was employed.

Assessments
Test Organism Observations
The number of fronds present in each test and control culture was recorded on days 0, 3, 5 and 7 along with observations on frond size, appearance, root length and number of colonies present.
In addition the dry weight of the fronds in each control and treatment group was determined on day 7. At the start of the test six replicate samples of fronds identical to those used to inoculate the test vessels were taken and the dry weight determined. At the end of the test the dry weight of colonies from each control and test vessel was determined by blotting the colonies dry and drying at 60 °C to constant weight.
Water Quality Criteria
The pH of each control and test flask was recorded on Day 0 and Day 7. The temperature and light intensity in the incubator were recorded daily.
Verification of Test Concentrations
Samples were taken from the control and 100% v/v saturated solution test group from the freshly prepared bulk test preparation on Day 0 and from the pooled replicates on Day 7 for quantitative analysis. All samples were stored frozen prior to analysis. Duplicate samples were taken at each occasion and stored frozen for further analysis if necessary.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Key result
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
> 0.092 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
Fron Number
Key result
Duration:
7 d
Dose descriptor:
NOEC
Effect conc.:
0.092 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
Fron Number
Key result
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
> 0.092 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
Dry Weight
Key result
Duration:
7 d
Dose descriptor:
NOEC
Effect conc.:
0.092 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
Dry Weight
Key result
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
> 0.092 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: Yield
Remarks:
Frond Number
Key result
Duration:
7 d
Dose descriptor:
NOEC
Effect conc.:
0.092 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: Yield
Remarks:
Frond Number
Key result
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
> 0.092 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: Yield
Remarks:
Dry Weight
Key result
Duration:
7 d
Dose descriptor:
NOEC
Effect conc.:
0.092 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: Yield
Remarks:
Dry Weight
Details on results:
Range-finding Test
The frond counts and percentage inhibition of growth values from the exposure of Lemna minor to the test item during the range-finding test are given in Table 1.
The results showed no significant effect on growth at the test concentration of 1.0, 10 and 100% v/v saturated solution.
Based on this information a single test concentration of six replicates, of 100% v/v saturated solution was selected for the definitive test. This experimental design conforms to a “limit test” to confirm that at the highest attainable test concentration, no effect on growth was observed.
Chemical analysis of the test preparations on Days 0, 3 and 7 showed measured test concentrations of less than the limit of quantification (LOQ) of the analytical method employed were obtained which was determined to be 0.095 mg/L. This does not infer that no test item was present, just that any which was, was at a concentration of less than 0.095 mg/L.

Definitive Test
Verification of Test Concentrations
Chemical analysis of the freshly prepared 100% v/v saturated solution test preparation on Day 0 showed a measured test concentration of 0.18 mg/L was obtained. Analysis of the 100% v/v saturated solution test preparation on Day 7 showed a decline in measured test concentration to less than the limit of quantification (LOQ) of the analytical method employed which was determined to be 0.095 mg/L.
Given this decline in measured test concentration it was considered justifiable to base the results on the geometric mean measured test concentration in order to give a “worst case” analysis of the data. The geometric mean measured test concentration was determined to be 0.092 mg/L.

Validation Criteria
The following data show that the doubling time of the control cultures was 1.92 days in line with the OECD Guideline that states the doubling time should be less than 2.5 days:
Mean frond number in control cultures at day 0 : 12
Mean frond number in control cultures at day 7 : 93

Growth Data Based on Frond Number
Numbers of fronds in each flask in the definitive test are reported in Table 2. Average specific growth rates, yields and percentage inhibition values are presented in Table 3.
Accordingly the following results based on inhibition of average specific growth rate and yield were determined from the frond number data:

Average Specific Growth Rate
ErC10 (frond number) = >0.092 mg/L
ErC20 (frond number) = >0.092 mg/L
ErC50 (frond number) = >0.092 mg/L
Where:
ErCx = the test concentration that reduced average specific growth rate by x%.
Statistical analysis of the average specific growth rate data was carried out for the control and 0.092 mg/L test concentration using a Student’s t-test incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981). There were no statistically significant differences between the control and 0.092 mg/L test concentration (P0.05) and therefore the "No Observed Effect Concentration" (NOEC) in terms of inhibition of average specific growth rates calculated from frond numbers was 0.092 mg/L.

Yield
EyC10 (frond number) = >0.092 mg/L
EyC20 (frond number) = >0.092 mg/L
EyC50 (frond number) = >0.092 mg/L
Where:
EyCx = the test concentration that reduced yield by x%.
Statistical analysis of the yield data was carried out for the control and 0.092 mg/L test concentration as above. There were no statistically significant differences between the control and 0.092 mg/L test concentration (P>0.05) and therefore the "No Observed Effect Concentration" (NOEC) in terms of inhibition of yield calculated from frond numbers was 0.092 mg/L.

Growth Data Based on Dry Weight
The dry weight of Lemna minor in each flask in the definitive test is reported in Table 4. Average specific growth rates, yield and percentage inhibition values are presented in Table 5.
Accordingly the following results based on inhibition of average specific growth rate and yield were determined from the dry weight data:

Average Specific Growth Rate
ErC10 (dry weight) = >0.092 mg/L
ErC20 (dry weight) = >0.092 mg/L
ErC50 (dry weight) = >0.092 mg/L
Where:
ErCx = the test concentration that reduced average specific growth rate by x%.
Statistical analysis of the average specific growth rate data was carried out for the control and 0.092 mg/L test concentration as above. There were no statistically significant differences between the control and 0.092 mg/L test concentration (P0.05) and therefore the "No Observed Effect Concentration" (NOEC) in terms of inhibition of average specific growth rate calculated from dry weight was 0.092 mg/L.

Yield
EyC10 (dry weight) = >0.092 mg/L
EyC20 (dry weight) = >0.092 mg/L
EyC50 (dry weight) = >0.092 mg/L
Where:
EyCx = the test concentration that reduced yield by x%.
Statistical analysis of the yield data was carried out for the control and 0.092 mg/L test concentration as in Section 6.2.4. There were no statistically significant differences between the control and 0.092 mg/L test concentration (P>0.05) and therefore the "No Observed Effect Concentration" (NOEC) in terms of inhibition of yield calculated from dry weight was 0.092 mg/L.

Observations
All test and control cultures were inspected on days 0, 3, 5 and 7.

Water Quality Criteria
The pH values of each test and control flask were in the ragne 6.7 - 9.4. Temperature was maintained at 24 ± 1 ºC throughout the test.
Results with reference substance (positive control):
A positive control (Envigo Study Number MM01PC) used 3,5-dichlorophenol as the reference item at concentrations of 0.625, 1.25, 2.5, 5.0 and 10 mg/L.
Exposure conditions and data evaluation for the positive control were similar to those in the definitive test.
Exposure of Lemna minor to the reference item gave the following results:
Response Variable Measurement Variable EC50 (mg/L) 95% Confidence Limits No Observed Effect Concentration (NOEC) (mg/L) Lowest Observed Effect Concentration (LOEC) (mg/L)
Average Specific Growth Rate Frond Number 3.4 3.1-3.8 0.625 1.25
Dry Weight 3.0 2.7-3.2 0.625 1.25
Yield Frond Number 1.8 1.6-2.2 0.625 1.25
Dry Weight 1.4 1.2-1.7 0.625 1.25
The results from the positive control with 3,5-dichlorophenol were within the normal ranges for this reference item.

Table 1            Frond Numbers and Percentage Inhibition of Growth from the Range-finding Test

Nominal Concentration

(% v/v Saturated Solution)

Number of Fronds

Average Specific Growth Rate

Yield

Day 0

Day 3

Day 5

Day 7

(0 – 7 Day)

% Inhibition

(0 – 7 Day)

% Inhibition

Control

R1

12

23

47

95

0.296

-

83

-

R2

12

24

44

87

0.283

75

Mean

12

24

46

91

0.290

79

1.0

R1

12

24

45

90

0.288

3

78

6

R2

12

26

47

81

0.273

69

Mean

12

25

46

86

0.281

74

10

R1

12

25

43

89

0.286

4

77

8

R2

12

26

40

80

0.271

68

Mean

12

26

42

85

0.279

73

100

R1

12

24

44

99

0.301

[6]

87

[14]

R2

12

23

50

105

0.310

93

Mean

12

24

47

102

0.306

90

R1– R2= Replicates 1 and 2

[Increase in growth compared to controls]

Table 2            Frond Numbers from the Definitive Test

Nominal Concentration

(% v/v Saturated Solution)

Number of Fronds

Day 0

Day 3

Day 5

Day 7

Control

R1

12

18

39

75

R2

12

22

40

94

R3

12

23

49

82

R4

12

21

49

111

R4

12

21

39

76

R6

12

25

49

121

Mean

12

22

44

93

100

R1

12

25

46

103

R2

12

22

43

95

R3

12

26

51

116

R4

12

25

45

93

R5

12

23

50

119

R6

12

23

40

94

Mean

12

24

46

103

Table 3            Inhibition of Average Specific Growth Rate and Yield Based on Frond Numbers

Nominal Concentration
(% v/v Saturated Solution)

Average Specific Growth Rate

Yield

(0 – 7 Day)

% Inhibition

(0 – 7 Day)

% Inhibition

Control

Mean

0.291

-

81

-

SD

0.029

19

100

Mean

0.307

[5]

91

[12]

SD

0.016

12

 

Table 4            Dry Weights from the Definitive Test

Nominal Concentration

(% v/v Saturated Solution)

Dry Weight (mg)

Day 0*

Day 7

Control

R1

 

8.1

R2

 

9.3

R3

 

7.9

R4

 

13.0

R5

 

6.0

R6

 

13.6

Mean

1.0

9.7

100

R1

 

9.9

R2

 

10.0

R3

 

13.7

R4

 

8.0

R5

 

12.5

R6

 

11.6

Mean

1.0

11.0

 

Table 5            Inhibition of Average Specific Growth Rate and Yield Based on Dry Weight

Nominal Concentration
(% v/v Saturated Solution)

Average Specific Growth Rate

Yield

(0 – 7 Day)

% Inhibition

(0 – 7 Day)

% Inhibition

Control

Mean

0.318

-

8.7

-

SD

0.045

3.0

100

Mean

0.340

[7]

10.0

[15]

SD

0.028

2.1

R1– R6= Replicates 1 to 6

SD– Standard Deviation

[Increase in growth compared to controls]

*Mean value determined from 6 replicate weighings

R1– R6= Replicates 1 to 6

SD– Standard Deviation

[Increase in growth as compared to the controls]

Validity criteria fulfilled:
yes
Conclusions:
The effect of the test item on the growth of Lemna minor has been investigated over a 7-Day period and based on the geometric mean measured test concentration showed Average Specific Growth Rate and Yield with respect to both Frond Number and Dry weight as EC50 > 0.092 mg/l and NOEC for the same endpoints was noted to be 0.092 mg/l.
This study showed that there were no toxic effects at saturation
Executive summary:

A study was performed to assess the effect of the test item on the growth of the freshwater plant Lemna minor. The method followed that described in the OECD Guideline No. 221 “Lemna sp.Growth Inhibition Test (March 2006)”.

Methods

Preliminary solubility work conducted indicated that it was not possible to obtain a testable solution of the test item using traditional methods of preparation e.g. ultrasonication and high shear mixing.

A preliminary media preparation trial indicated that a dissolved test item concentration of approximately 0.63 mg/L was obtained from a saturated solution method of preparation indicating this to be the limit of water solubility of this item under test conditions.

Following a preliminary range-finding test and initial experiment,Lemna minorwas exposed to an aqueous solution of the test item at a nominal concentration of 100% v/v saturated solution (six replicate flasks) for a period of 7 days, under constant illumination at a temperature of 24 ± 1°C. The test item solutions were prepared by stirring an excess (50 mg/L) of test item in culture medium using a propeller stirrer at approximately 1500 rpm for 48 hours. After the stirring period any undissolved test item was removed by centrifugation at 40000gfor 30 minutes to produce a 100% v/v saturated solution of the test item.

The number of fronds in each control and treatment group was recorded on days 0, 3, 5 and 7 along with observations on plant development.

Results

Chemical analysis of the freshly prepared 100% v/v saturated solution test preparation on Day 0 showed a measured test concentration of 0.18 mg/L was obtained. Analysis of the 100% v/v saturated solution test preparation on Day 7 showed a decline in measured test concentration to less than the limit of quantification (LOQ) of the analytical method employed which was determined to be 0.095 mg/L. 

Given this decline in measured test concentration it was considered justifiable to base the results on the geometric mean measured test concentration in order to give a “worst case” analysis of the data. The geometric mean measured test concentration was determined to be 0.092 mg/L. 

Exposure of Lemna minor to the test item based on the geometric mean measured test concentration gave the following results:

Response Variable

Measurement Variable

EC50(mg/L)

No Observed Effect Concentration (NOEC) (mg/L)

Average Specific Growth Rate

Frond Number

>0.092

0.092

Dry Weight

>0.092

0.092

Yield

Frond Number

>0.092

0.092

Dry Weight

>0.092

0.092

 

This study showed that there were no toxic effects at saturation.

Description of key information

The effect of the test item on the growth of Lemna minor has been investigated over a 7-Day period and based on the geometric mean measured test concentration showed Average Specific Growth Rate and Yield with respect to both Frond Number and Dry weight as EC50 > 0.092 mg/l and NOEC for the same endpoints was noted to be 0.092 mg/l. This study showed that there were no toxic effects at saturation.

Key value for chemical safety assessment

EC50 for freshwater plants:
0.092 mg/L
EC10 or NOEC for freshwater plants:
0.092 mg/L

Additional information

A study was performed to assess the effect of the test item on the growth of the freshwater plant Lemna minor. The method followed that described in the OECD Guideline No. 221 “Lemnasp.Growth Inhibition Test (March 2006)”. The effect of the test item on the growth of Lemna minor has been investigated over a 7-Day period and based on the geometric mean measured test concentration showed Average Specific Growth Rate and Yield with respect to both Frond Number and Dry weight as EC50 > 0.092 mg/l and NOEC for the same endpoints was noted to be 0.092 mg/l. This study showed that there were no toxic effects at saturation.