7,7-dimethyl-3-oxa-6-azaoctan-1-ol

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The definitive test was conducted between 26 May 2004 and 16 June 2004. The last data collected for this study was on 23 June 2004.

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.

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Deviations:

no

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: 2nd December 2002, Date of Signature: 13th February 2003

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 1.0, 3.2, 10, 32 and 100 mg/l.
- 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 Day 0 and of the expired test preparationson Days 2, 5, 7, 9, 12, 14, 16, 19 and 21.
- Sample storage conditions before analysis: Duplicate samples were taken and stored at approximately -20°C for further analysis if necessary.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
For the purpose of the definitive test the test material was dissolved directly in reconstituted water.
The following test concentrations were assigned to the definitive test: 1.0, 3.2, 10, 32 and 100 mg/l.
The stock solution and each prepared concentration were inverted several times to ensure adequate mixing and homogeneity.
The concentration and stability of the test material in the test preparations were verified by chemical analysis on Days 0, 2, 5, 7, 9, 12, 14, 16, 19 and 21 (see attached background material for results).

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water flea.
- Strain/clone: Not reported.
- Source: Derived from in-house laboratory cultures.
- Age of parental stock: The test was carried out using 1st instar Daphnia Magna.
- Feeding during test
- Food type: Each culture was fed daily with a suspension of algae (Chlorella sp).



ACCLIMATION
Not applicable.



METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES:
Culture conditions ensured that reproduction was by parthenogenesis. Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old. These young were removed from the cultures and used for testing.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Post exposure observation period:

Not applicable.

Hardness:

The water hardness was observed to be in the range 230 to 258 mg/l as CaCO3 in the control and the highest surviving test group throughout the test.

Test temperature:

Temperature was maintained at approximately 21°C throughout the test.

pH:

pH was recorded before and after each test media renewal.
A range of 7.9 - 9.9 was recorded. Concentration dependent differences in pH were observed throughout the test.

Dissolved oxygen:

Dissolved oxygen concentrations were recorded before and after each test media renewal.
A range of 8.0 - 8.7 mg O2/l was recorded.
No treatment related differences for oxygen concentration observed.

Salinity:

Not reported.

Nominal and measured concentrations:

Nominal concentrations: 1, 3.2, 10, 32 and 100 mg/l.
Analysis of the test preparations throughout the test showed the measured test concentrations to be within the accepted limits of 80% to 120% of the nominal values with four exceptions, which showed measured test concentrations of 65%, 70%, 71% and 75% of the nominal values. In these cases duplicate frozen samples were analysed. The results of these analyses showed measured test concentrations of 68%, 82%, 100% and 125% of nominal, indicating that the true dosed concentrations were likely to have been within the accepted values of 80% to 120% of nominal. It was therefore considered appropriate to base the results of the test on the nominal test concentrations only.
Please see attached background material (Appendix 1) for the verification of test concentrations results.

Details on test conditions:

TEST SYSTEM
- Test vessel:Glass flasks
- Type (delete if not applicable): closed (covered with glass lid).
- Material, size, headspace, fill volume: 150 ml glass flasks.
- Aeration: The test vessels were not aerated.
- Type of flow-through (e.g. peristaltic or proportional diluter): Not applicable.
- Renewal rate of test solution (frequency/flow rate):The test preparations were renewed 3 times per week on Days 2, 5, 7, 9, 12, 14, 16 and 19.
- For each concentration a single daphnid was placed in the test preparation. For each test and control group ten replicate test vessels were prepared.


TEST MEDIUM / WATER PARAMETERS
The reconstituted water used for the definitive test was the same as that used to maintain the stock animals.
The reconstituted water had a pH of 7.8 +/- 0.2 adjusted (if necessary) with NaOH or HCl and was aerated until the dissolved oxygen concentration was approximately air-saturation value.
The reconsititued water had an approximate theoretical total hardness of 250 mg/l as CaC03.


OTHER TEST CONDITIONS
- Adjustment of pH: pH of reconstituted water adjusted if necessary.
- Photoperiod: The flasks were maintained at approx 21°C with a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods for 21 days.
- Light intensity: 508 to 574 lux.


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
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.
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 (i.e. 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.

At the end of the test, the length of each surviving parent animal was determined.
VEHICLE CONTROL PERFORMED: no


RANGE-FINDING STUDY
Not conducted. Based on data supplied the test concentrations were assigned for the definitive test.

Reference substance (positive control):

no

Duration:

14 d

Dose descriptor:

EC50

Effect conc.:

36 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 25-51 mg/l

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

18 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 10-32 mg/l

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

18 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: 95% Confidence Limits of 10-32 mg/l

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

immobilisation

Remarks:

in the parental Daphnia generation (P1)

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

immobilisation

Remarks:

in the parental Daphnia generation (P1)

Details on results:

The observations for each test and control group are summarised in Tables 1 to 7. The total cumulative production of live young is given in Table 8 and the number of live young produced per adult is shown in Table 9 (see attached background material for all tables).

Results with reference substance (positive control):

Not appliacable.

Reported statistics and error estimates:

Please see below.

Validation Criteria:

The following validation criteria were achieved during the test.

	Required	Actual
a) Control mortality	≤ 20%	Zero
b) Dissolved oxygen	> 3 mg O2/l	> 8.0 mg O2/l
c) pH (control group)	Deviation ≤ 1.5	0.3
d) mean number of live young per surviving adult (control group)	≥ 60 afer 21 days	71
e) Coefficient of variation for control group	≤ 25%	4.8%

Lethal Effects on the Parental Generation (P₁):

Mortality (immobilisation) occured predominantly at the highest test concentrations of 32 and 100 mg/l resulting in 100% mortality by days 16 and 13 respectively indicating a prolonged toxic effect attributable to exposure of Daphnia Magna to the test material.

No mortalities occurred at the 1.0, 3.2 and 10 mg/test concentrations throughout the test.

The following EC50 (immobilisation) values based on nominal test concentrations were calculated by the trimmed Spearman-Karber method at 14 days and the geometric mean method at 21 days:

Time	EC50 (mg/l)	95% Confidence Limits
24 hours	>100	-
48 hours	>100	-
96 hours	>100	-
7 days	>100	-
14 days	36	25 – 51
21 days	18	10 -32*

*Concentrations resulting in 0 and 100% immobilisation respectively

It was not possible to calculate the EC50 value until Day 11 of the test, as less than 50% mortalities were observed at the highest concentration of 100 mg/l.

Sub-lethal Effects on the Parental Generation (P₁)

There was a significant effect on size and colour of the daphnids in that all of the adult daphnids at the 100 mg/l test concentration were markedly paler in colour than the control animals from days 3 to 5. Thereafter all of the surviving daphnids were markedly smaller and paler in colour than the control animals prior to observing 100% mortalities by Day 13.

There was a significant effect on the colour of 10% to 100% of the surviving daphnids at the 32 mg/l test concentration from Day 7 until Day 16 when 100% mortality was observed.

The daphnids at the remaining test concentrations were observed to be the same size and colour as the control animals.

After 21 days there were no statistically significant differences between the control and the 1.0, 3.2 and 10 mg/l test groups in terms of the number of live young produced per adult. The 32 and 100 mg/l test groups were not included in the statistical analysis as exposure to the test material eliminated all the daphnids prior to Day 21 of the test.

The EC50 (reproduction) value calculated by the geometric mean method on Day 21, based on nominal test concentrations was 18 mg/l.

After 21 days the length of each surviving adult was determined, the results of which are given in Appendix 3 (attached background material). The results show that there were no statistically significant differences (P ≥ 0.05) between the control and the 1.0, 3.2 and 10 mg/l test groups in terms of length of the daphnids after 21 days exposure to the test material. The 32 and 100 mg/l test groups were not included in this analysis as exposure to the test material eliminated all the daphnids prior to Day 21 of the test.

Effects on the Filial Generation (F₁)

Information on the effects of the test material on the F₁ generation is limited, since, by study design, the young are removed soon after liberation from the brood pouch. However, an assessment made at each media renewal showed the "filial" daphnids produced by all the test groups were in the same general condition as the young produced by the controls over the duration of the test.

Young were first produced in the control test group on Day 7 of the test.

Numbers of unhatched eggs and dead young were low in all control and treatment groups surviving to maturation.

Lowest Observed Effect Concentration:

The LOEC was 32 mg/l as 100% mortality was observed at this test group.

No Observed Effect Concentration:

The NOEC was 10 mg/l as there were no mortalities (immobilisation) observed in the parental generation (P₁) and there were no significant differences in terms of the number of live young produced per adult when compared to the control after 21 days.

Validity criteria fulfilled:

yes

Conclusions:

Exposure to Daphnia magna to the test material result in significant mortalities at the test concentrations of 32 and 100 mg/l resulting in 100% mortalities by Days 16 and 13 respectively.

The 14 and 21-Day EC50 (immobilisation) values, based on nominal test concentrations, for the parental Daphnia generation (P1) were calculated to be 36 and 18 mg/l with 95% confidence limits of 25-51 mg/l and 10-32* mg/l respectively.

The 21-Day EC50 (reproduction) based on nominal test concentrations was 18 mg/l with 95% confidence limits of 10 - 32* mg/l.

The "Lowest Observed Effect Concentration" (LOEC) and the "No Observed Effect Concentration" NOEC based on nominal test concentrations were 32 and 10 mg/l respectively.

* Concentrations resulting in 0 and 100% immobilisation respectively.

Executive summary:

Introduction.

A study was performed to assess the effect of the test material on the reproduction ofDaphnia magnaover 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 data supplied by the Sponsor,Daphnia magnawere exposed (10 replicates of a single daphnid per group) to an aqueous solution of the test material over a range of test concentrations of 1.0, 3.2, 10, 32 and 100 mg/l for a period of 21 days. The test solutions were renewed 3 times per week. The numbers of live and deadDaphniaand young daphnids (live and dead) were determined daily. TheDaphniawere fed daily with an algal suspension.

Results.

The 14 and 21-Day EC₅₀(immobilisation) values, based on nominal test concentrations, for the parentalDaphniageneration (P₁) were calculated to be 36 mg/l and 18 g/l with 95% confidence limits of 25 -51 mg/l and 10 -32 mg/l* respectively.

The 21 -Day EC₅₀(reproduction) value based on nominal test concentrations was calculated to be 18 mg/l with 95% confidence limits of 10 -32 mg/l. Care should be taken in interpretation of this value as no effects of exposure to the test material were shown at the test concentration of 10 mg/l, which was the No Observed Effect Concentration and 100% mortality was observed at the test concentration of 32 mg/l prior to termination of the test after 21 days.

The "Lowest Observed Effect Concentration" was considered to be 32 mg/l on the basis that at this test concentration significant mortalities (immobilisation) were observed in the parental generation (P₁) over the duration of the test.

Analysis of the test preparations throughout the test showed the measured test concentrations to be within the accepted limits of 80% to 120% of the nominal values with four exceptions, which showed measured test concentrations of 65%, 70%, 71% and 75% of the nominal values. In these cases duplicate frozen samples were analysed. The results of these analyses showed measured test concentrations of 68%, 82%, 100% and 125% of nominal, indicating that the true dosed concentrations were likely to have been within the accepted values of 80% to 120% of nominal. It was therefore considered appropriate to base the results of the test on the nominal test concentrations only.

* Concentrations resulting in 0 and 100% mortality respectively.

The "No Observed Effect Concentration" was considered to be 10 mg/l in the basis that at this test concentration there were no mortalities (immobilisation) observed in the parental generation (P₁) and there were no significant differences (P ≥0.05) between the control and the 10 mg/l test group in terms of numbers of live young produced per adult by Day 21.

 

Description of key information

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).
The 14 and 21-Day EC50 (immobilisation) values, based on nominal test concentrations, for the parental Daphnia generation (P1) were calculated to be 36 and 18 mg/l with 95% confidence limits of 25-51 mg/l and 10-32* mg/l respectively.
The 21-Day EC50 (reproduction) based on nominal test concentrations was 18 mg/l with 95% confidence limits of 10 - 32* mg/l.
The "Lowest Observed Effect Concentration" (LOEC) and the "No Observed Effect Concentration" NOEC based on nominal test concentrations were    32 and 10 mg/l respectively.
* Concentrations resulting in 0 and 100% immobilisation respectively.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

10 mg/L