4,4'-[(1,10-dioxodecane-1,10-diyl)bis(oxy)]bis(2,2,6,6-tetramethylpiperidine-1-oxidanyl)

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1997

Report date:

1997

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Version / remarks:

The study design was based on the OECD guideline No. 202: "Daphnia sp., Acute Immobilisation Test and Reproduction Test', Adopted April 4, 1984, extended with recommendations and supplements which are included in the Draft OECD guideline 202 Part II prepared in 1996. This design was also in agreement with the EEC Draft guideline XI/681/86: 'Prolonged Toxicity Study with Daphnia magna: Effects on Reproduction"; Version 4.

Deviations:

not specified

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

1,10-bis(2,2,6,6-tetramethyl-1-piperidinyloxy)-1,10-dioxodecane

EC Number:

417-440-8

EC Name:

1,10-bis(2,2,6,6-tetramethyl-1-piperidinyloxy)-1,10-dioxodecane

Cas Number:

2516-92-9

Molecular formula:

C28 H50 N2 O6

IUPAC Name:

bis(1-hydroxy-2,2,6,6-tetramethylpiperidin-4-yl) decanedioate

Details on test material:

Batch: CXA681901

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: During the study samples were taken from the control and the saturated concentration of CXA 5415 (100 %).
- Sampling method:
Frequency: Prior to the start of the test (day -1) to check the efficiency of the generator column. From the fresh solutions at the beginning of the test and on nominal days 6, 8, 14 and 20, and from the 48-hour old solutions on day 2 and on day 22.
Volume: 300 to 500 ml.
- Sample storage conditions before analysis: Samples not analysed on the day of sampling were stored in a deep-freeze until analysis.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
The test substance is a powder with a very low solubility in water (<0.0005 g/l). The standard test procedures for the present chronic test require generation of test solutions which contain completely dissolved test substance concentrations. The testing of concentrations above the maximum solubility will disturb the test system or induce physical damage to the test organisms owing to the presence of an undissolved fraction of test substance. Therefore, the test solution consisted of a saturated solution. This saturated solution was generated from a generator consisting of test substance adsorbed on glass pearls. The procedure of preparation of the column is as follows. 2 grams of the test substance was dissolved in acetone. An weighed amount of ca. 40 grams of glass pearls was added to this solution, and mixed carefully. Thereafter, the solvent was evaporated completely at approximately 30''C, using a rotary evaporator. The loaded carrier material was stored at room temperature in the dark prior to further use. M7-medium (without vitamins) was recirculated through the generator to provide for a saturated solution. The recirculation periods between renewal of test solutions were ca. 48 hours. Glass wool was used as a barrier between the generator and the reservoir vessel. At each renewal, the necessary volumes of saturated solution were taken from the reservoir and the same volume of freshly prepared M7-medium (without vitamins) was added to the reservoir. After addition of vitamins, the saturated solution was divided over the different vessels. M7-medium (with vitamins) was used as such in the control.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: common water flea
- Strain/clone: not specified
- Source: laboratory culture
- Age of parental stock : maximum 4 weeks
- Feeding during test:
Daily, a volume of 0.25 ml of a Chlorella pyrenoidosa suspension with a cell density of 2*10^8 cells/ml was added to each vessel containing ca. 50 ml of test solution as feed for the daphnids i.e. a ration per organism of about 50*10^6 cells/day (corresponding with ca. 0.5 mg C/Daphnia/day).

ACCLIMATION
not specified

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

22 d

Test conditions

Test temperature:

20.6-21.4°C

pH:

8.0-8.4

Dissolved oxygen:

>8 mg/L

Nominal and measured concentrations:

The test groups consisted of:
b. Negative control group: Untreated test medium.
a. CXA 5415 treated group: The saturated solution of CXA 5415 produced by a column generator;

Details on test conditions:

TEST SYSTEM
- Test vessel: All glass; Volume: 50 ml ( 5 x 0 3.7 cm), covered with a perspex plate.
- Aeration:
- Type of flow-through (e.g. peristaltic or proportional diluter):
- Renewal rate of test solution (frequency/flow rate):
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 20
- No. of vessels per control (replicates): 20
- Biomass loading rate: 1 daphnia / 50 ml

TEST MEDIUM / WATER PARAMETERS
M7

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hours photoperiod daily, 600 lux.
- Light intensity: not specified

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :

TEST CONCENTRATIONS
The test groups consisted of:
b. Negative control group: Untreated test medium.
a. CXA 5415 treated group: The saturated solution of CXA 5415 produced by a column generator;

- Justification for using less concentrations than requested by guideline: because the test substance was hard to dissolve, only one test concentration could be prepared (maximum dissolved concentration).

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

22 d

Dose descriptor:

LOEC

Effect conc.:

> 0.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Effect concentrations 2

Duration:

22 d

Dose descriptor:

LOEC

Effect conc.:

> 0.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

immobilisation

Details on results:

The mortality of the parental daphnids in the controls did not exceed 20%. The average cumulative number of young per female in the controls after 21 days was > 60.

Comparison of the reproduction data of the parental organisms exposed to the test medium recirculated through the CXA 5415 treated column generator with those of the control group showed no distinction in the reproductive capacity of Daphnia magna. During the total exposure period of 22 days, no significant (>10%) immobilisation (including mortality) of newborn young was observed at any of the test concentrations. Further, no biologically significant numbers of unhatched eggs were observed during the reproduction phase.

The results of this study should be interpreted considering the results of concentration analyses. Although the detection efficiency of the analytical method had been increased, the analytical program produced detectable concentrations in only two of the six samples taken from the column generator during the test period. Hence, it was not possible for the analytical program to confirm that a stable saturated concentration was reached using the column generator. Simulation tests were performed using the same design for the column generator, and detectable concentrations were found in samples taken in these simulation tests. However, again quantification was hampered by poor recoveries. This lack of analytical confirmation of actual test concentrations is mainly a consequence of the hydrophobic property of CXA 5415 and indicates that the solubility of CXA 5415 in test medium appears to be much lower than 0.5 mg/l.

Reported statistics and error estimates:

no statistics were necessary.

Any other information on results incl. tables

Measured concentrations of CXA 5415 in water samples:

No detectable concentrations were found in most samples. However, incidentally detectable amounts of CXA 6415 were found. Recovery experiments showed that the recovery for method of sample treatment prior to analyses was depending on concentration and varied between 20 and 70%.

Hence, the lack of detectable concentrations was at least partly caused by poor efficiency of recovery. A simulation test was performed and detectable concentrations were found in samples taken. However, again quantification was hampered by poor recoveries.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

This study investigated the effects of exposure to a saturated solution of CXA 5415 on survival and reproductive performance in Daphnia magna during 22 days of exposure. Prolonged exposure of Daphnia magna to test medium which was recirculated through a generator column with CXA 5415 adsorbed to glass pearls did not induce any effect on the survival or reproduction of these organisms.

Despite the fact that concentrations of CXA 5415 could not be detected analytically in several samples from the column generator, the results indicate that the generator produced a saturated solution with CXA 5415 concentrations varying below 0.05 mg/l. Hence, the solubility of CXA 5415 in test medium appeared to be more than a factor of 10 lower than 0.5 mg/l.

The present test assessed the possible risks of prolonged exposure of crustaceans in a case of high loading of the aquatic environment by CXA 5415. The results show that in such a case no concentrations of CXA 5415 will be present in the water phase that have any effect on the survival or reproduction of these organisms.

Executive summary:

This study investigated the effects of CXA 5415 on survival and reproductive performance in Daphnia magna during 22 days of exposure.

The study design was based on the OECD guideline No. 202: "Daphnia sp., Acute Immobilisation Test and Reproduction Test', Adopted April 4, 1984, extended with recommendations and supplements which are included in the Draft OECD guideline 202 Part II prepared in 1996. This design was also in agreement with the EEC Draft guideline XI/681/86: 'Prolonged Toxicity Study with Daphnia magna: Effects on Reproduction"; Version 4.

According to data supplied by the sponsor, the water solubility of CXA 5415 is < 0.5 mg/l. The standard test procedures for the present chronic test required generation of test solutions which contained completely dissolved test substance concentrations. Therefore, the test solution was a saturated solution generated using a column generator with test substance adsorbed to glass pearls. M7-medium (without vitamins) was recirculated through the generator to provide for a saturated solution. The experiment started with 20 vessels per test group, each vessel containing one neonate (<24h old) Daphnia magna in 50 ml test media. Duration of the study was 22 days and the test solutions were renewed every 48 hours. At the start of the test and every workday, the condition of the parental daphnia was recorded and during the reproduction phase the number of living offspring, immobile young and number of unhatched eggs were recorded.

During the study samples were taken from freshly prepared solutions on days 0, 8, 14 and 20 and from the 48-hour old solutions on day 2 and day 22. The analytical program produced detectable concentrations in two of these six samples. Recovery experiments showed that the recovery for method of sample treatment prior to analyses was depending on concentration and varied between 20 and 70%. Detectable concentrations were found in samples taken

in simulation tests with the column generator, but again quantification was hampered by poor recoveries. In conclusion, despite the fact that concentrations of CXA 5415 could not be detected analytically in several samples from the column generator, the results indicated that the generator produced a saturated solution with CXA 5415 concentrations varying below 0.05 mg/l. Hence, the solubility of CXA 5415 in test medium appeared to be more than a factor of 10 lower than 0.5 mg/l.

During the 22-day exposure period, the mortality rate in the blank control did not exceed 20%. Mortality rate in the saturated test group was 25%. This was not considered as a test substance related effect. Comparison of both test groups showed that no effect was observed on the reproduction of the parental Daphnia.

The present test assessed the possible risks of prolonged exposure of crustaceans in a case of high loading of the aquatic environment by CXA 5415. The results show that in such a case no concentrations of CXA 5415 will be present in the water phase that have any significant effect on the survival or reproduction of these organisms.