1-Propanaminium, N,N,N-trimethyl-3-(octadecyloxy)-, chloride (1:1)

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:

28 November 2007 to 18 February 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.20 (Daphnia magna Reproduction Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of Inspection: 21 August 2007 Date of Signature: 15 October 2007

Test material

Test material information

Constituent 1

Reference substance name:

trimethyl[3-(octadecyloxy)propyl]azanium chloride

EC Number:

700-414-8

Cas Number:

23328-71-4

Molecular formula:

C24H52ClNO

IUPAC Name:

trimethyl[3-(octadecyloxy)propyl]azanium chloride

Details on test material:

- Name of test material (as cited in study report): 3-Octadecyloxypropyl-N,N,N-trimethylammonium chloride
- Molecular formula (if other than submission substance): Not applicable
- Molecular weight (if other than submission substance): Not applicable
- Smiles notation (if other than submission substance): Not applicable
- InChl (if other than submission substance): Not applicable
- Structural formula attached as image file (if other than submission substance): Not applicable
- Substance type: white solid
- Physical state: solid
- Analytical purity: Not reported
- Impurities (identity and concentrations): Not reported
- Composition of test material, percentage of components: Not reported
- Isomers composition: Not reported
- Purity test date: Not reported
- Lot/batch No.: Exp. I-070518
- Expiration date of the lot/batch: Not reported
- Radiochemical purity (if radiolabelling): Not applicable
- Specific activity (if radiolabelling): Not applicable
- Locations of the label (if radiolabelling): Not applicable
- Expiration date of radiochemical substance (if radiolabelling): Not applicable
- Stability under test conditions: Please see Table 1 and 2. A range of preliminary test samples was prepared, analysed initially and then after storage in sealed glass vessels at ambient temperature in light and dark conditions for approximately 48 hours and 72 hours (equivalent to the maximum periods of media renewal). In addition test samples were tested for stability without prior mixing (sonication) of the test sample bottles to assess for losses due to adsorption and/or insolubility. Further portions of test samples were analysed following the addition of algal cells to assess the effects of algae on the stability of test material in test medium.
The test samples have been shown to be unstable in the test medium in light and dark conditions. However, based on the chemical structure this was deemed not to be due to hydrolysis.
The unsonicated stability vessels showed no significant evidence of insolubility or adherence to glass.
The presence of algal cells was considered to have a significant effect on the stability of the test material in the test medium.
- Storage condition of test material: room temperature in the dark.
- Other: Not reported.

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
Not reported.

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0, 0.00017, 0.00054, 0.0017, 0.0054 and 0.017 mg/l (nominal).
- Sampling method: Water samples were taken from the control and each surviving test group (replicates pooled) for quantitative analysis. Samples of the fresh test preparations were taken on Days 0, 2, 4, 7, 9, 11, 14, 16 and 18 and of the expired test preparations on Days 2, 4, 7, 9, 11, 14, 16, 18 and 21. Water samples were also taken from the stock solutions used to prepare the test series on Days 0, 2, 4, 7, 9, 11, 14, 16 and 18.
- Sample storage conditions before analysis: Duplicate samples were taken and stored at approximately -20°C for further analysis if necessary.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
An amount of test material (100 mg) was dissolved in dechlorinated tap water with the aid of ultrasonication for approximately 50 minutes and the volume adjusted to 1 litre to give a 100 mg/l stock solution from which serial dilutions were prepared in dechlorinated tap water to give further stock solutions of 10, 1.0, 0.10 and 0.010 mg/l. Aliquots (34 and 108 ml) of the 0.010 mg/l stock solution were each separately dispersed in a final volume of 2 litres of dechlorinated tap water to give the 0.00017 and 0.00054 mg/l test concentrations respectively. Aliquots (34, 108 and 340 ml) of the 0.10 mg/l stock solution were each separately dispersed in a final volume of 2 litres of dechlorinated tap water to give the 0.0017, 0.0054 and 0.017 mg/l test concentrations respectively.
Each stock solution and prepared concentration was inverted several times to ensure adequate mixing and homogeneity.
The concentration and stability of the test material in the stock solutions and the test preparations were verified by chemical analysis on Days 0 (fresh media), 2, 4, 7, 9, 11, 14, 16, 18 (fresh and old media) and 21 (old media).
- Differential loading: Not reported.
- Controls: The control group was maintained under identical conditions but not exposed to the test material.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Not applicable.
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): Not applicable.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): The freshly prepared test media were observed to be clear, colourless solutions, whereas the old test media were observed to be green tinged due to the presence of algal cells used as feed for the daphnids.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water flea.
- Strain/clone: Not reported.
- Source: In-house laboratory cultures.
- Age of parental stock (mean and range, SD):1st instar.
- Feeding during test: Each culture was fed daily with a suspension of algae (Chlorella sp.). The diet is considered not to contain any contaminant that would affect the integrity or outcome of the study. Each daphnid received approximately 5 to 10 µl of a unicellular algal culture (Chlorella sp.), daily. Feeding was at a level of approximately 0.1 to 0.2 mg carbon/daphnid/day, dependent on the age and size of the animals. Equal amounts of food were given to each daphnid.

ACCLIMATION
Not applicable.

QUARANTINE (wild caught)
Not applicable.

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES:
The adult Daphnia were transferred to fresh media by wide-bore pipette before the contents of each vessel were passed through a fine mesh. Young daphnids (live and dead) and any unhatched eggs were collected on the mesh and counted using a stereo microscope before being discarded.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Post exposure observation period:

Not applicable.

Test conditions

Hardness:

The water hardness was observed to be in the range 128 to 166 mg/l as CaCO3 in the control and the highest surviving test group throughout the test.

Test temperature:

Temperature was maintained at approximately 20°C throughout the test.

pH:

7.9 - 8.4.

Dissolved oxygen:

7.9 - 9.6 mg O2/l.

Salinity:

Not reported.

Nominal and measured concentrations:

0.00017, 0.00054, 0.0017, 0.0054 and 0.017 mg/l (nominal).
0.00019, 0.00028, 0.00066, 0.0019 and 0.0062 mg/l (measured).

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): closed. Covered with a plastic lid to reduce evaporation.
- Material, size, headspace, fill volume: 150 ml glass flasks.
- Aeration: The test vessels were not aerated. The diluent water only was aerated prior to use.
- Type of flow-through (e.g. peristaltic or proportional diluter): Not applicable.
- Renewal rate of test solution (frequency/flow rate): 3 times per week on Days 2, 4, 7, 9, 11, 14, 16 and 18.
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- No. of vessels per vehicle control (replicates): Not applicable.
- Biomass loading rate: Not reported.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
Laboratory tap water was dechlorinated by passage through an activated carbon filter (Purite Series 500) and partly softened (Elga Nimbus 1248D Duplex Water Softener) giving water with a total hardness of approximately by 140 mg/l as CaCO3. After dechlorination and softening the water was passed through a series of computer controlled plate heat exchangers to achieve the required temperature.
- Total organic carbon: (Average) 1.429 mg/l
- Particulate matter: Not reported.
- Metals: (Average)
Al <10.596 ug/l
Cd <0.213 µg/l
Cr <0.551 ug/l
Cu <0.021 mg/l
Fe <28.038 µg/l
Pb <1.113 µg/l
Hg <0.013 µg/l
Mn <1.723 ug/l
Ni <2.325 µg/l

- Pesticides: (Average) 0.019 µg/l
- Chlorine: (Average) 0.274 mg/l
- Alkalinity: (Average) pH 7.655
- Ca/mg ratio: Not reported.
- Conductivity: 403.577 µS/cm at 20°C
- Salinity: Not reported.
- Culture medium different from test medium: No.
- Intervals of water quality measurement: Not reported.

OTHER TEST CONDITIONS
- Adjustment of pH: No.
- Photoperiod: Photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods for 21 days.
- Light intensity: 479 to 534 lux.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Mortality: On a daily basis the numbers of live and dead of the "Parental" (P1) generation, the numbers of live and dead "Filial" (F1) Daphnia and the number of discarded unhatched eggs were counted. An assessment was also made of the general condition and size of the parental Daphnia as compared with the controls.
- Fertility: The number of Daphnia with eggs or young in the brood pouch was determined daily. Young daphnids were considered to be dead if no sign of movement was apparent during microscopic examination. Adult Daphnia which were unable to swim for approximately 15 seconds after gentle agitation (ie. immobile), were considered to be dead. An immobilisation criterion for the young daphnids was considered to be inappropriate due to the large numbers of off-spring produced in the flasks.
- Growth: At the end of the test, the length of each surviving parent animal was determined.

VEHICLE CONTROL PERFORMED:
Not applicable

RANGE-FINDING STUDY
- Test concentrations: Not applicable.
- Results used to determine the conditions for the definitive study:
Based on the results of an acute toxicity test to 1st instar Daphnia magna (Safepharm Laboratories Project Number: 0140/1413 - see section 6.1.3) the following test concentrations were assigned to the definitive test: 0.00017, 0.00054, 0.0017, 0.0054 and 0.017 mg/l.

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

0.001 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

immobilisation

Remarks:

for the parental Daphnia generation (P1)

Remarks on result:

other: 95% confidence limits of 0.00088 – 0.0012 mg/l

Effect concentrations 2

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

0.001 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: 95% confidence limits of 0.00047 – 0.00085 mg/l

Effect concentrations 3

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

0.001 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mortality

Effect concentrations 4

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mortality

Effect concentrations 5

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

0.003 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

immobilisation

Remarks:

for the parental Daphnia generation (P1)

Remarks on result:

other: 95% confidence limits of 0.0022 – 0.0034 mg/l

Effect concentrations 6

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

0.002 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: 95% confidence limits of 0.0011 – 0.0022 mg/l

Effect concentrations 7

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

0.002 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Effect concentrations 8

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.001 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Details on results:

- Mortality of parent animals:
Mortality (immobilisation) occurred predominantly at the highest test concentrations of 0.0054 and 0.017 mg/l resulting in 100% mortality by Day 21 indicating a prolonged toxic effect attributable to exposure of Daphnia magna to the test material.
One mortality was also observed at the test concentration of 0.00054 mg/l on Day 12. However, statistical analysis of the mortality data using the corrected chi-squared statistic (see Appendix 3) (Breslow and Day 1980) showed that the observed mortality in the 0.0054 mg/l test group was not significantly different (P≥0.05) when compared to the control group.
No mortalities occurred at the 0.00017 and 0.0017 mg/l test concentrations throughout the test.
The EC50 (immobilisation) value based on nominal test concentrations was calculated by the trimmed Spearman-Karber method (Hamilton et al 1977) at 21 days (Table 3).
It was not possible to calculate the EC50 value until Day 21 of the test since zero or less than 50% mortalities were observed.

- No. of offspring produced per day per female:
After 21 days there were no statistically significant differences between the control, 0.00017 and 0.00054 mg/l test groups in terms of the number of live young produced per adult. The 0.0017 mg/l test group showed a statistically significant difference from the control and the remaining test groups after 21 days in terms of producing fewer numbers of live young per adult. The 0.0054 and 0.017 mg/l test groups data were not included in statistical analysis as exposure to the test material eliminated all the daphnids by Day 21 of the test.

- Body length and weight of parent animals:
The results showed that there were no statistically significant differences (P< 0.05) between the control and the 0.00017 and 0.00054 mg/l test groups in terms of length of the daphnids after 21 days exposure to the test material However the 0.0017 mg/l test group was observed to be statistically (P<0.05) smaller than the control group. The 0.0054 and 0.017 mg/l test groups data were not included in this analysis as exposure to the test material eliminated all the daphnids by Day 21 of the test.

- Type and number of morphological abnormalities:
There was a significant effect on colour of the daphnids in that 33, 40, 100 and 80% of the surviving daphnids on Days 9, 15, 16 and 19 at the test concentration of 0.017 mg/l were markedly paler in colour than the control animals.
The daphnids at the remaining test concentrations were observed to be the same size and colour as the control animals with the exception of 11% and 22% of the surviving daphnids in the 0.0054 mg/l test group on Days 18 and 19 which were observed to be pale compared to the controls from Day19 until observed dead on Day 20.

- Time to first brood release or time to hatch
An assessment made at each media renewal showed the "filial" daphnids produced by the all the test groups were in the same general condition as the young produced by the controls over the duration of the test with the exception of on Day 12 when some of the young produced the 0.00054, 0.0017 and 0.0054 mg/l test groups and the majority of the young produced by 0.017 mg/l test group were observed to be dead and on Day 19 when the majority of the young produced by the 0.017 mg/l test group were observed to be dead.
Young were first produced in the control test group on Day 8 of the test.
Numbers of unhatched eggs and dead young were low in all control and treatment groups surviving to maturation.

- Egg development time: Not examined.
- Brood size: Please see Table 4 attached.
- Time to sexual maturity: Not examined.
- Type and magnitude of biochemical changes: Not reported.
- Effect concentrations exceeding solubility of substance in test medium: The freshly prepared test media were observed to be clear, colourless solutions, whereas the old test media were observed to be green tinged due to the presence of algal cells used as feed for the daphnids.
- Others
Verification of Test Concentrations
Analysis of the freshly prepared stock solution samples used to prepare the test series showed the majority of measured concentrations to be within 80% – 120% of nominal with the exception of 8 samples which showed measured concentrations of 127% to 285% of nominal. Examination of the data did not reveal why these samples were out of specification.
Analysis of the freshly prepared test concentrations (see Appendix 2) showed the majority of measured concentrations to range from 49% to 140% of nominal with the exception of the lowest test concentration which showed measured concentrations to range from less than the LOQ to 340% of nominal. These low and variable measured test concentrations were considered to be due to possible absorption to the glassware used in the preparation of the test series, given that the test material was a quaternary ammonium compound. The variation in the lowest test concentration was also considered to be due to the very low test concentration and the associated variability of analysis at such low levels and that small variations in actual concentration gave rise to correspondingly large variation in the percentage of nominal values and was not considered to have affected the outcome or integrity of the study as the 0.00017 mg/l test concentration was below the No Observed Effect Concentration (NOEC).
The measured concentrations of the old or expired test media showed measured values to range from less than the LOQ to 873%. The variable results were considered to be due to reasons given above. In addition, inspection of the data showed an overall trend for a decline in measured concentration over each media renewal period. However, this was also considered to be due to instability of the test material in water and was in line with the preliminary stability analysis performed.
Duplicate frozen samples were analysed for some of the concentrations that were out of specification. The results showed an improved general trend compared to the original results (majority within the 80% to 120% acceptable limits) confirming the majority of the analysis of the original high measured test concentrations to be erroneous.
Overall, the analysis of the test concentrations showed a concentration dependent increase in the measured concentration with increase in nominal concentration throughout the duration of the test in both the fresh and old test concentration samples. However, given the general trend for decline in measured concentration over each media renewal period it was therefore considered justifiable to base the results on the time-weighted mean measured test concentrations of the test media to give a "worst case" analysis of the data (see Table 5).
The 21-Day EC50 (immobilisation) value based on the time-weighted mean measured test concentrations of the test media was calculated to be 0.0010 mg/l with 95% confidence limits of 0.00088 - 0.0012 mg/l.
The 21-Day EC50 (reproduction) value based on the time-weighted mean measured test concentrations of the test media was calculated to be 0.00063 mg/l with 95% confidence limits of 0.00047 - 0.00085 mg/l.
The “Lowest Observed Effect Concentration” (LOEC) and “No Observed Effect Concentration” (NOEC) based on the time-weighted mean measured test concentrations were 0.00066 and 0.00028 mg/l respectively.
The use of time-weighted mean measured test concentration to calculate the results did significantly affect the results of the test.

Results with reference substance (positive control):

Not applicable.

Reported statistics and error estimates:

Not reported.

Any other information on results incl. tables

Validation Criteria

The following validation criteria were achieved during the test:

	Required	Actual
a        Control mortality	<=20%	0%
b       Dissolved oxygen	> 3 mg O2/l	>7.9
c        pH (control group)	deviation<=1.5	0.4
d       Mean number of live young per surviving adult (control group)	³60 after 21 days	104
e        Coefficient of variation for control group	<=25%	24.9%

Table 3 EC₅₀(immobilisation) values

Time	EC50 (mg/l)	95% Confidence Limits (mg/l)
24 hours	>0.017	-
48 hours	>0.017	-
96 hours	>0.017	-
7 days	>0.017	-
14 days	>0.017	-
21 days	0.0027	0.0022 - 0.0034*

*Concentrations resulting in 0 and 100% immobilisation respectively

Table 5  Nominal -  Time Weighted Mean Measured concentrations.

Nominal Test Concentration (mg/l)	Time Weighted Mean Measured Concentration (mg/l)	% of nominal
0.00017	0.00019	109
0.00054	0.00028	51
0.0017	0.00066	39
0.0054	0.0019	35
0.017	0.0062	37

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Exposure of Daphnia magna to the test material resulted in significant mortalities at the test concentrations of 0.0054 and 0.017 mg/l (0.0019 and 0.0062 mg/l based on the time-weighted mean measured test concentrations of the test media) resulting in 100% mortalities by Day 21.
The 21-Day EC50 (immobilisation) value, based on nominal test concentrations, for the parental Daphnia generation (P1) were calculated to be 0.0027 mg/l (0.0010 mg/l based on the time-weighted mean measured test concentrations of the test media) with 95% confidence limits of 0.0022 – 0.0034 mg/l respectively (0.00088 – 0.0012 mg/l based on the time-weighted mean measured test concentrations of the test media).
The 21-Day EC50 (reproduction) based on nominal test concentrations was 0.0015 mg/l (0.00063 mg/l based on the time-weighted mean measured test concentrations of the test media) with 95% confidence limits of 0.0011 – 0.0022 mg/l (0.00047 – 0.00085 mg/l based on the time-weighted mean measured test concentrations of the test media).
The "Lowest Observed Effect Concentration" (LOEC) and the "No Observed Effect Concentration" (NOEC) based on nominal test concentrations were 0.0017 and 0.00054 mg/l respectively (0.00066 and 0.00028 mg/l based on the time-weighted mean measured test concentrations of the test media respectively).

Executive summary:

Introduction.

A study was performed to assess the effect of the test material on the reproduction of Daphnia magna over a 21-Day period. The method followed that described in the OECD Guidelines for Testing of Chemicals No 211 (1998) "Daphnia magna, Reproduction Test", referenced as Method C.20 of Commission Directive 2001/59/EC (which constitutes Annex V of Council Directive 67/548/EEC).

Methods.

Based on the results of an acute toxicity test (Safepharm Laboratories Project No: 0140/1413), Daphnia magna were exposed (10 replicates of a single daphnid per group) to an aqueous solution of the test material over a range of test concentrations of 0.00017, 0.00054, 0.0017, 0.0054 and 0.017 mg/l for a period of 21 days. The test solutions were renewed 3 times per week. The numbers of live and dead adult Daphnia and young daphnids (live and dead) were determined daily. The Daphnia were fed daily with an algal suspension.

Results.

The 21-Day EC₅₀ (immobilisation) value, based on nominal test concentrations, for the parental Daphnia generation (P₁) was calculated to be 0.0027 mg/l with 95% confidence limits of and 0.0022 – 0.0034 mg/l respectively.

The 21-Day EC₅₀(reproduction) value, based on nominal test concentrations, was calculated to be 0.0015 mg/l with 95% confidence limits of 0.0011 – 0.0022 mg/l.

The "Lowest Observed Effect Concentration" was considered to be 0.0017 mg/l on the basis that at this test concentration significantly fewer live young per adult (P<0.05) were produced when compared to the control by Day 21.

The "No Observed Effect Concentration" was considered to be 0.00054 mg/l on the basis that at this test concentration there were no significant mortalities (immobilisation) observed in the parental generation (P₁) and that there were no significant differences (P³0.05) between the control and the 0.00054 mg/l test group in terms of numbers of live young produced per adult by Day 21.

Analysis of the freshly prepared stock solution samples used to prepare the test series showed the majority of measured concentrations to be within 80% – 120% of nominal with the exception of 8 samples which showed measured concentrations of 127% to 285% of nominal. Examination of the data did not reveal why these samples were out of specification.

Analysis of the freshly prepared test concentrations showed the majority of measured concentrations to range from 49% to 140% of nominal with the exception of the lowest test concentration which showed measured concentrations to range from less than the limit of quantitation (LOQ) to 340% of nominal. These low and variable measured test concentrations were considered to be due to possible absorption to the glassware used in the preparation of the test series, given that the test material was a quaternary ammonium compound. The variation in the lowest test concentration was also considered to be due to the very low test concentration and the associated variability of analysis at such low levels and that small variations in actual concentration gave rise to correspondingly large variation in the percentage of nominal values and was not considered to have affected the outcome or integrity of the study as the 0.00017 mg/l test concentration was below the No Observed Effect Concentration (NOEC).

The measured concentrations of the old or expired test media showed measured values to range from less than the LOQ to 873%. The variable results were considered to be due to reasons given above. In addition, inspection of the data showed an overall trend for a decline in measured concentration over each media renewal period. However, this was also considered to be due to instability of the test material in water and was in line with the preliminary stability analysis performed.

Duplicate frozen samples were analysed for some of the concentrations that were out of specification. The results showed an improved general trend compared to the original results (majority within the 80% to 120% allowable limits) confirming the majority of the analysis of the original high measured test concentrations to be erroneous.

Overall, the analysis of the test concentrations showed a concentration dependent increase in the measured concentration with increase in nominal concentration throughout the duration of the test in both the fresh and old test concentration samples. However, given the general trend for decline in measured concentration over each media renewal period, it was considered justifiable to base the results on the time-weighted mean measured test concentrations of the test media to give a "worst case" analysis of the data.

The 21-Day EC₅₀ (immobilisation) value based on the time-weighted mean measured test concentrations of the test media, for the parental Daphnia generation (P₁) were calculated to be 0.0010 mg/l with 95% confidence limits of 0.00088 – 0.0012 mg/l.

The 21-Day EC₅₀ (reproduction) value based on the time-weighted mean measured test concentrations of the test media was calculated to be 0.00063 mg/l with 95% confidence limits of 0.00047 – 0.00085 mg/l.

The “Lowest-Observed Effect Concentration” and the “No Observed Effect Concentration” based on the time-weighted mean measured test concentrations of the test media were 0.00066 and 0.00028 mg/l respectively.