Registration Dossier

Ecotoxicological information

Long-term toxicity to aquatic invertebrates

Administrative data

Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 08 June 2000 to 18 Dec 2000
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: OECD Guideline study with acceptable deviations

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report Date:
2000

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Deviations:
yes
Remarks:
1) Daphnids were not maintained individually (one/test vessel). 2) Carbon content of test medium and mgC/daphnia/day from feeding are not mentionned in the study report. 3) Results were expressed in term of arithmetic means instead of time-weighted mean.
Qualifier:
equivalent or similar to
Guideline:
other: OECD Guideline 202 (Part II)
Deviations:
yes
Remarks:
Daphnia were fed during the test
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): ST 08 C 99
- Substance type: pure active substance
- Analytical purity: 99.5 %
- Isomers composition: ca. 10-12 % cis- and 88-90 % trans- isomers
- Lot/batch No.: F84912-08D
- Expiration date of the lot/batch: 24 May 2002
- Stability under test conditions: stable at pH 7 (t1/2> 1 year) , stability at pH9: t1/2 = 7.8 days. Not volatile in the test conditions
- Storage condition of test material: not mentionned in study report
- Other: test material is moderately soluble (water solubility > 500 mg/l @ 20°C), has a low Vapour pressure (< 0.0001 Pa) and a logPow < 3
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
Not Applicable

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Not mentionned in the study report
- Concentrations: all concentrations on day 0 (fresh), day 2 (old solution), day 7 and 14 (old and fresh solution) and on day 21 (old solution)
- Sampling method: 10.0 ml sample of each new solution was collected from the parent test solution. At sample points for "old" solutions, the replicate test chambers (A, B, C and D) were composited and a 10.0-mL sample was collected from each treatment composite. Control and ST 08 C 99 fortified QC samples were also analyzed at each sample point.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
Testing material was soluble in the test conditions (Water solubility of 561 mg/l according to OECD 105 Guideline)
- Method: test concentrations were prepared on a weight/volume basis.

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna
- Source: ABC laboratories in-house culture, Missouri- USA
- Age of parental stock (mean and range, SD): The adult daphnids that produced the neonates were cultured in a temperature-controlled water bath at approximately 20°C. During the holding period, the adult daphnids received a diet of algae (Selenastrum capricornutum and/or Ankistrodesmus falcatus) supplemented by a prepared artificial invertebrate food. The adult daphnids used in the definitive test were cultured for approximately 19 days and were considered acceptable with no signs of stress, disease, or physical damage. The adult daphnids were isolated from the culture and transferred to dilution water <24 hours prior to the beginning of the definitive test.
- Feeding during test
- Food type: Selenastrum capricornutum and Ankistrodesmus falcatus and suspension of an artificial invertebrate diet
- Amount: Daphnids were fed a diet consisting of a one-milliliter volume of a 1 x 100'000'000 cells/ml algal suspension (Selenastrum capricornutum and Ankistrodesmus falcatus) once each day and supplemented once each day with 0.5 ml of a 5 mg/ml suspension of an artificial invertebrate diet. This diet yielded a final algal concentration of at least 250'000 cells/ml/day and 6.25 µg suspended solids/ml/day of the artificial invertebrate diet during the definitive test.
- Frequency: daily


ACCLIMATION
The adult daphnids that produced the neonates were cultured in a temperature-controlled water bath at approximately 20°C. During the holding period, the adult daphnids received a diet of algae (Selenastrum capricornutum and/or Ankistrodesmus falcatus) supplemented by a prepared artificial invertebrate food. The adult daphnids used in the definitive test were cultured for approximately 19 days and were considered acceptable with no signs of stress, disease, or physical damage. The adult daphnids were isolated from the culture and transferred to dilution water <24 hours prior to the beginning of the definitive test.
- Acclimation conditions (same as test or not): yes


METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES:
Daphnids were tranferred daily to clean test chambers which contained freshly prepared test solution. The animals were initially released or transferred into the appropriate test chambers within thirty minutes of preparing fresh test solutions. Transfer of daphnids from "old" solutions (approximately 24 hours old) to "new" solutions (freshly prepared) was accomplished using a large-bore glass pipet. Tranfers began with the control replicates and then proceeded from the lowest treatment to the highest treatment. After the first brood release, only the adult daphnids were transferred to new test solutions. The neonates produced between each renewal and at test termination were collected, enumerated, and discarded.

Study design

Test type:
other: static renewal conditions
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Post exposure observation period:
Not Applicable

Test conditions

Hardness:
At beginning (day 0): 144 & 146 mg/l CaCO3, at termination (day 21): 164 & 160 mg/l CaCO3
Test temperature:
20 ± 1 °C (19.3° to 20.4°C) (See Temperature, Oxygen and pH measurements under "attached background material")
pH:
8.17 to 8.56 (See Temperature, Oxygen and pH measurements under "attached background material")
Dissolved oxygen:
6.84 to 9.32 mg/O2 corresponding to 79 to 107% of saturation (See Temperature, Oxygen and pH measurements under "attached background material")
Salinity:
Not Applicable
Nominal and measured concentrations:
See Table 1 "Measured Concentrations of the test material " in Remarks on results incl. tables and figures"
Concentrations in the exposure system were determined on freshly prepared "new" solutions on day 0, 7 and 14 and in "old" solutions collected from the exposure system on days 2, 7, 14 and 21. The measured concentrations of ST 08 C 99 in new solutions ranged from 0.123 to 16.8 mg/l
and were 94 to 108% of the nominal concentrations. The measured concentrations of ST 08 C 99 in old solutions ranged from 0.0558 to 15.3 mg/l
and were 43 to 96% of the nominal concentrations. The overall mean measured concentrations (mean of new and old measured concentrations) of ST 08 C 99 ranged from 0.1 to 16.04 mg/l and were 74 to 101% of the nominal concentrations.
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Material, size, headspace, fill volume: Glass jars covered with plastic petri dish covers. Each test chamber measured approximately 9 cm in diameter by 17 cm in height. Solution depth was approximately 7 cm, for a volume of approximately 400 mL.
- Aeration: none
- Renewal rate of test solution (frequency/flow rate): Daphnids were tranferred daily to clean test chambers which contained freshly prepared test solution. (Transfer of daphnids from "old" solutions (approximately 24 hours old) to "new" solutions (freshly prepared) was accomplished using a large-bore glass pipet).
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The dilution water was a laboratory blended freshwater that originated from a deep well. A portion of this well water was passed though a reverse osmosis system and then blended back with raw well water to achieve a hardness between 130 to 160 mg/L as CaC03. The dilution water was biologically aged and passed through a cartridge filter and ultraviolet sterilizer before use in the test.
- Total organic carbon: Not mentionned in the study report
- Particulate matter: Not mentionned in the study report
- Metals: in accordance with historical range
- Pesticides: in accordance with historical range
- Chlorine: Not mentionned in the study report
- Alkalinity: 140 - 160 mg/l CaCO3
- Conductivity: Day 0: 318 µS, Day 21: 307 µS
- Culture medium different from test medium: no
- Intervals of water quality measurement: at least once a year

OTHER TEST CONDITIONS
- Adjustment of pH: none
- Photoperiod: 16 hours light / 8 hours dark with 30 min simulated dawn and dusk periods
- Light intensity: 520.5 - 602.8 lux


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Immobilization was defined as lack of movement of appendages or postabdomen within 15 seconds after gentle agitation of the test chamber or gentle disturbance of the daphnid itself. Daphnids were observed daily and any mortality and/or sublethal effects were recorded.


RANGE-FINDING STUDY
- Test concentrations: First range study (static): 0 (control), 0.10, 1.0, 10, and 100 mg ST 08 C 99/l. Second range study (flow-through): 0 (control), 0.063, 0.13, 025, 0.50, and 1.0 mg ST 08 C 99/l. First test attempt (flow-trough): 0 (control), 0.13, 025, 0.50, 1.0, and 2.0 mg ST 08 C 99/

- Results used to determine the conditions for the definitive study:
Prior to the initiation of the definitive test, two range-finding tests and a definitive test attempt were conducted. The first rangefinding test was conducted as a static test from June 8 to 10, 2000, using nominal concentrations of 0 (control), 0.10, 1.0, 10, and 100 mg ST 08 C 99/l. After 48 hours of exposure, the percent immobilization was 100% at concentrations ≥ 10 mg ST 08 C 99/l. No immobilization occurred in the control or test solutions ≤ 1.0 mg/L. Based upon these results, the nominal test concentrations were selected for the second range-finding test.
The second range-finding test was conducted as a flow-through exposure from June 16 to 30, 2000, using nominal concentrations of 0 (control), 0.063, 0.13, 025, 0.50, and 1.0 mg ST 08 C 99/l in duplicate replication. After 14 days of exposure, there was no significant mortality as compared to the control. Reproduction began on day 7 in all control and test solution replicates. The total number of instar produced was 1743, 1695, 1665, 1556, 1309, and 1132 (or 10.9, 11.2, 10.5, 9.73, 8.18, and 7.53 young per adult reproductive day) for control, 0.063, 0.13, 025, 0.50, and 1.0 mg ST 08 C 99/l, respectively. Based upon these results, the nominal test concentrations were selected for the definitive test attempt.
A definitive test attempt was conducted as a flow-through exposure from August 2 to 23, 2000, using nominal concentrations of 0 (control), 0.13, 0.25, 0.50, 1.0, and 2.0 mg ST 08 C 99/l in quadruplicate replication. After 21 days of exposure, there was no signifIcant mortality as compared to the control. Reproduction began on day 7 in all control and test solution replicates. The total number of instar produced was 7026, 7307, 6708, 6967, 7325, and 5986 (or 12.0, 13.1, 11.5, 11.6, 13.1, and 10.0 young per adult reproductive day) for control, 0.13, 025, 0.50, 1.0, and 2.0 mg ST 08 C 99/L, respectively. There was no signifIcant difference in dry weight or lengths of the surviving adult daphnids at day 21. The concentrations of the test solutions were measured on days 0, 2, 7, 9, 14, 16, and 21. The mean measured concentrations were
Reference substance (positive control):
no

Results and discussion

Effect concentrationsopen allclose all
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
0.79 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
1.73 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
1.73 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
3.72 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 16.1 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
0.732 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
1.86 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks on result:
other: 0.62-2.43
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
2.01 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: 1.64-2.28
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
3.05 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
other: length
Remarks on result:
other: 1.01-3.51
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
> 1.82 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
other: Dry weight
Details on results:
- Mortality of parent animals: After 21 days of exposure, immobility of D. magna exposed to ST 08 C 99 ranged from 1% at 0.790 mg/L to 100% at 7.82 and 16.1 mg/l. Immobility in the dilution water control was 0%. A statistically significant increase in immobility (as compared to the control) was detected for the three highest ST 08 C 99 treatments tested (3.72, 7.82 and 16.1 mg/L) as well as the lowest treatment (0.0968 mg/L). The significant increase in immobility detected for the 0.0968 mg/L treatment was not considered to be biologically significant since the next four higher treatments (0.199, 0.400, 0.790, and 1.73 mg/L) showed no significant increase (for more details, refer to "Immobilization of daphnids" in "background attached material")
- No. of offspring produced per day per female: Not Applicable as measurements were performed in quadruplicate of 10 daphnids. The mean
numbers of young per reproductive day or "YAD" ranged from 0 at 7.82 and 16.1 mg/l to 8.92 at 0.0968 mg/l. The number of young produced per adult reproductive day was significantly lower than the control for the 1.73 mg/l treatment. The 3.72, 7.82, and 16.1 mg/l treatments were omitted from the analysis due to significant effects on survival at these concentrations. (for more details, see "Number of Youngs produced & Day of first brood" in "background attached material")
- Body length and weight of parent animals: Mean length of the dilution water control animals was 4.2 mm. Mean lengths of daphnids in the ST 08 C 99 treatments ranged from 3.5 mm at 3.72 mg/L to 4.2 mm at 0.968 and 0.400 mg/l. The mean total length of daphnids was significantly less than those of the control for the 1.73 mg/l treatment. The 3.72, 7.82, and 16.1 mg/l treatment were omitted from the analysis due to significant effects on survival at these concentrations. Mean dry weight of the dilution water control daphnids was 0.56 mg. Mean dry weights of surviving daphnids in the ST 08 C 99 treatments ranged from 0.45 mg at 3.72 mg/l to 0.67 mg at 0.0968 mg/l. The mean weight of daphnids in treatments ≤ 1.73 mg/L were not significantly less than those of the control. The 3.72, 7.82, and 16.1 mg/l treatments were omitted from the analysis due to significant effects on survival at these concentration. (for more details, see "Lengths and Mean dry weight of first generation" in "background attached material").
- Type and number of morphological abnormalities: not mentionned in the study report
- Type and number of behavioural abnormalities: not mentionned in the study report
- Time to first brood release or time to hatch: young were first observed on day 7 or 8 in all replicates of the control and for the five lowest tested concentrations (mean measured concentrations: 0.0968, 0.199, 0.400 & 0.79, 1.73 mg/l). In the next concentration (3.72 mg/ml) young were first observed on day 8 for one of the four replicates and on day 9, 12 and 12 for the remaining three replicates. For higher tested concentrations (7.82 and 16.1 mg/l), no young were produced.
- Brood size: number of young/adult/reproductive day or "YAD" was determined (see above)
- Effect concentrations exceeding solubility of substance in test medium: none
Results with reference substance (positive control):
Not Applicable
Reported statistics and error estimates:
See "Statistical Analysis" in "Remarks on results including tables and figures" and the report "Hedione _Chronic toxicity study to Daphnia magna_final_10032014.pdf" in attached documents.

Any other information on results incl. tables

Analytical monitoring results

 

Table1: Measured Concentrations of the test material

 

Nominal concentration (mg/l)

Sampleday

 

Control

0.13

0.25

0.50

1.0

2.0

4.0

8.0

16

16

0

(fresh)

<MQL

0.127

0.262

0.506

1.00

2.16

4.15

8.12

16.8

17.1

2

(old)

<MQL

0.104

0.221

0.440

0.890

1.89

3.56

7.51

15.3

NS

7

(fresh)

<MQL

0.127

0.258

0.523

0.990

2.09

4.15

NS

NS

16.3

7

(old)

<MQL

0.0558

0.134

0.273

0.559

1.49

3.65

NS

NS

NS

14

(new)

<MQL

0.123

0.241

0.493

0.942

1.96

3.86

NS

NS

15.0

14

(old)

<MQL

0.0564

0.109

0.235

0.460

1.09

2.91

NS

NS

NS

21

(old)

<MQL

0.0846

0.167

0.332

0.647

1.40

3.76

NS

NS

NS

 

Meanconcentration

-

0.0968

0.199

0.400

0.790

1.73

3.72

7.82

16.1

16.1

% of nominal

-

74

80

80

79

87

93

98

101

101

 

MQL : Minimum QuantifiableLimit(0.009 mg/l)

NS : NotSampled

 

Table 2: measured concentrations of ST 08 C 99 in Quality Control Fortifications

Nominal QC Spike Concentration (mg/l)

Measured Concentrations as mg/l (% of nominal)

Day 0

Day 2

Day 7

Day 14

Day 21

0.126

0.129 (102)

0.129 (102)

0.129 (102)

0.128 (102)

0.131 (104)

20.1

22.7 (113)

22.3 (111)

22.8 (113)

20.7 (103)

21.5 (107)

 

Summary of Results

The effect of ST 08 C 99 on survival, reproduction, and growth (length and dry weight) of Daphnia magna following 21 days of exposure is summarized in the following table:

 

Mean measured Test Concentration TWM (mg/l)

Percent Survival (%)

Days to First Brood

Young/Adult Reproduction Day

Mean Total Length (mm)

Mean Dry Weight (g)

 

Control

 

100

 

7.25

 

8.54

 

4.17

0

.56

0.10

85*

7.25

8.92

4.23

0.67

0.21

95

7.50

8.13

4.14

0.63

0.42

95

7.25

8.20

4.17

0.63

0.83

98

7.25

8.09

4.15

0.61

1.82

95

7.75

7.96

4.07

0.55

3.74

15

10.25

1.87

3.51

0.45

7.81

0

a

a

a

a

16.04

0

b

b

b

b

 

a Complete mortality was observed prior to day 5

b Complete mortality was observed prior to day 4

*This valuewas considered an aberrant effect since higher test concentrations (0.199, 0.400, 0.790, and 1.73 mg/l) did not produce significant effects

 

Detailed results and corresponding data tables:(Temperature/O2/pH measurements, Immobilization of daphnids, Number of Young Produced & Day of first brood, Lengths and mean dry weight),see “attached background material.

 

Statistical analysis

Experimental units, on which observations or measurements were made, were the replicated beakers. All statistical analyses were performed using SAS for Windows, Release 6.12. Inferences of statistical significance were based upon a P ≤ 0.05 unless otherwise noted.

Survival, expressed as a percentage, was calculated by subtracting cumulative immobility at the end of the test from the initial number of daphnids present at test initiation. Using the count of dead/alive summed over the four replicates, separate contingency tables were constructed andpairwisecomparisons between the control and each of the ST 08 C 99 treatments were performed using a Fisher's exact test. A Hochberg adjustment was used to control the experiment-wise error rate for the Fisher's test at the same alpha level.

 

Reproductive success variables consisted of time to first brood (days) and young per adult reproductive day (YAD). For each individual replicate, the time to first brood was the day on which neonates were first observed.

The YAD was calculated by the following equation:

YAD= total number of neonates/total number of adult reproduction days

 

Adult reproduction days were based on the number of days adultdaphnidswere reproducing and the number of adultdaphnidsalive on each day. The number of reproduction days was counted from the day neonate production was first observed in any chamber to the last day of the test. The value for adult reproduction days was calculated by multiplying the number of adultdaphnidsalive in each replicate for each reproduction day. Reproduction days either never accrued or stopped accruing for adults which died during the test.

 

Growth variables consisted of length and dry weight. The length analysis was performed on the mean adult length per replicate. The weight analysis was performed on the mean adult dry weight in each replicate.

Reproduction and growth data were analyzed using a one-way analysis of variance (ANOVA). A one-tailedDunnett'stest was conducted at the P ≤0.05 level of significance, with comparison to the control group. Prior to the Dunnett's test, a Shapiro-Wilk's test and a Levene's test for normality and homogeneity of variance over treatments at each day were conducted. Where the P values from the Shapiro-Wilk's and Levene's test were greater than 0.01, indicating normality and insignificant heterogeneity, the analysis was performed on the raw value. Where the P value was less than 0.01, a log transformation was used. Only treatment levels not shown to be affected statistically (P > 0.05) forsurvival wereincluded in the reproduction and growth data analysis.

 

Based upon the results of the above statistical analyses, the MATC geometric mean was calculated as follows:

Antilog of [(log NOEC + log LOEC)/2] = MATC geometric mean.

 

Statistical analysis of the mean measured ST 08 C 99 concentrations verses immobility data was performed using a SAS program that calculated the EC50 .statistic along with the 95% confidence limits using theprobitmethod and/or Trimmed Spearman-Karbermethod.

A further statistical method using the "ToxRat Professional" probit statistical tool was applied to determine the EC10 for each endpoint.

 

Applicant's summary and conclusion

Validity criteria fulfilled:
not applicable
Conclusions:
The most sensitive biological endpoints for Daphnia magna exposed for 21 days to ST 08 C 99 were reproduction (young produced per adult reproduction day) and growth (adult length). Therefore, the EC10 for reproduction was 1.86 mg/l. The EC10 for adultsurvival was 2.01 mg/l. The EC10 for length was 3.05 mg/l.
The EC10 for mean dry weight of adult daphnids at treatments > 1.82 mg/l.
Executive summary:

PURPOSE:

The objectives of this study were to determine the affect of ST O8 C 99 on survival, reproduction, and growth of the water flea, Daphnia magna,during a complete life cycle exposure under static renewal test conditions, secondarily, to establish the Maximum Acceptable Toxicant Concentration (MATC) limits of ST O8 C 99 for Daphnia magna based upon biological effect criteria of survival, reproduction, and growth, and thirdly, to determine a 48-hour EC50 (acute) concentration. This test was based on the OECD Guidelines for the Testing of Chemicals, Methods 202 (Part II) and 211"Daphnia sp., Acute Immobilization Test and Reproduction Test" and was supplemented by ABC Laboratories' standard operating procedures (SOP's). The results derived from this test satisfy data requirements specified by the OECD guideline with acceptable restrictions.

SUMMARY:

A 21-day static renewal toxicity test (i.e., test solutions were renewed by transfer ofdaphnidsto fresh test solution each day) was conducted at ABC Laboratories in Columbia, Missouri, to evaluate the effects of ST 08 C 99 on Daphnia magna.The criteria for effects were survival, reproduction, and growth (length and dry weight).

The definitive toxicity test consisted of a no-treatment group and eight groups exposed to ST 08 C 99. The following nominal concentrations were tested: 0.0 (no-treatment control), 0.13, 0.25, 0.50, 1.0, 2.0, 4.0, 8.0, and 16 mg ST 08 C 99/liter. The no-treatment control daphnids were exposed to dilution water only. All test solutions were prepared in dilution water which was ABC freshwater possessing a total hardness of l30 to 160 mg/L CaC03.

The definitive toxicity test was initiated with the addition of daphnids, less than 24 hours of age, into test chambers. The test chambers were glass jars containing 400 ml of test solution. There were four replicates per treatment with ten daphnids per replicate, which provided a total of forty daphnids per each treatment and control group at test initiation. The test chambers were randomly positioned in a temperature-controlled water bath that maintained the temperature of the test solutions between 19 and 20°C during the test.

Water samples for chemical analysis of each viable control and treatment were taken on days 0, 7, and 14 for "new" (freshly prepared) solutions and on days 2, 7, 14, and 21 for "old" (~24 hours old) solutions during the definitive test. All samples were analyzed by GC MSD following analytical methodology validated by ABC Laboratories, Inc. The mean measured concentrations of ST 08 C 99 for new solutions were 0.126, 0.254, 0.507, 0.977, 2.07, 4.05, 8.12, and 16.8 mg/l and 97 to 105% of the targeted nominal concentrations. The mean measured concentrations of ST 08 C 99 for old solutions were 0.0752, 0.158, 0.320, 0.649, 1.47, 3.47, 7.51, and 15.3 mg/l and 58 to 96% of the targeted nominal concentrations. The Time Weighted Mean measured concentrations of ST 08 C 99 for both new and old solutions were 0.10, 0.21, 0.42, 0.83, 1.82, 3.74, 7.81, and 16.04 mg/l and 74 to 101% of the targeted nominal concentrations. The test substance was considered to be soluble; however, the concentrations were not stable under the test conditions at levels below 4 mg/l, based upon the results of the definitive measured concentrations.

The test chambers were observed daily and any mortality and/or sublethal effects were recorded. Young were collected from the time of first brood release (day 7) and every following day including test termination. The collected young were enumerated and reproduction was evaluated by calculating the number of young produced per adult reproduction day. At test termination (day 21), the lengths and dry weights of the surviving adult daphnids were determined.

The effect of ST 08 C 99 on survival, reproduction, and growth (length and dry weight) of Daphnia magna following 21 days of exposure is summarized in the following table:

 

Mean measured Test Concentration TWA (mg/l)

Percent Survival (%)

Days to First Brood

Young/Adult Reproduction Day

Mean Total Length (mm)

Mean Dry Weight (g)

 

Control

 

100

 

7.25

 

8.54

 

4.17

0

.56

0.1

85*

7.25

8.92

4.23

0.67

0.21

95

7.50

8.13

4.14

0.63

0.42

95

7.25

8.20

4.17

0.63

0.83

98

7.25

8.09

4.15

0.61

1.82

95

7.75

7.96

4.07

0.55

3.74

15

10.25

1.87

3.51

0.45

7.81

0

a

a

a

a

16.04

0

b

b

b

b

 

a Complete mortality was observed prior to day 5

b Complete mortality was observed prior to day 4

*This value was considered an aberrant effect since higher test concentrations (0.182, 0.42, 0.83, and 1.82 mg/l) did not produce significant effects

 

Following 21 days of exposure, a significant effect on survival was detected for 0.1, 3.74, 7.81, and 16.04 mg/L, as compared to the control. The survival effect observed for 0.1 mg/l was considered an aberrant effect since higher test concentrations (0.1, 0.42, 0.83, and 1.82 mg/l) did not produce significant effects. Therefore, the no-observed-effect concentration (NOEC) and the lowest-observed-effect concentration (LOEC) for survival were 1.73 and 3.72 mg/L, respectively.

All individual control and ≤1.82 mg/l treatment replicates were observed to produce young on days 7 or 8. The number of young produced per adult reproduction day started to decrease from 1.82 mg/l, as compared to the control. The 7.81, and 16.04 mg/L treatments were omitted from statistical analysis due to observed significant survival effects. In a supporting document, attached to this dossier, The original statistical methods used by the authors were reviewed as the NOEC and LOEC values overlap an important regulatory threshold (with the NOEC just below 1 mg/l and the LOEC well above this value) and therefore another statistical method, ECx was also calculated before chosing a final chronic value for this study. The conclusion was that, due to the subjectivity of the dos selection of the NOEC and LOEC, the EC10 was a superior statistical tool to determine the endpoint value in this case. For all the chronic toxicity endpoints the EC10 was well above 1.0 and were therefore used to determine the final endpoint value for this study.

Therefore, the EC10 for reproduction was 1.86 mg/l.

The EC10 for adultsurvival was 2.01 mg/l.

The EC10 for length was 3.05 mg/l.

The EC10 for mean dry weight of adult daphnids at treatments > 1.82 mg/l.