Reaction products of neodecanoic acid and titanium (IV) isopropoxide

Long-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

other information

Study period:

09-Aug-2011 to 08-Sep-2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

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. Conclusive valid guideline study under GLP conditions.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

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:

swissmedic, decision: 19-November-2010

Test material

Test material information

Constituent 1

Reference substance name:

E0286P/040A

IUPAC Name:

E0286P/040A

Constituent 2

Reference substance name:

E0286P/040A/01

IUPAC Name:

E0286P/040A/01

Details on test material:

The test item and the following information concerning the test item were provided by the Sponsor:

Identity: E0286P/040A
Batch No.: E0286P/040A/01
Composition: 80% titanium salts and 20% neo-decanoic acid
Expiration Date (as given on the test item label): 31-Dec-2011
Storage Conditions (as provided by the Supplier): Keep container tightly closed. Keep container in a cool, well-ventilated area.
Storage Conditions (as handled at Harlan Laboratories): At room temperature at about 20 °C, in the dark.

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

For verification of the presence of the test item in the test media (based on the analysis of one test item component, i.e. neo-decanoic acid), duplicate samples were taken from the freshly prepared test media of each test item loading rate and the control at the first treatment period, at a treatment period in the second week and at one of the last treatment periods (Day 0, 7, and 16, respectively).

To determine the maintenance of neo-decanoic acid in the test media, stability samples were taken at the end of two test medium renewal periods of 48 hours (Days 2 and 9) and at the end of one renewal period of 72 hours (Day 19):

a) Samples with food and test animals were taken from the actual test by combining the contents of all replicate test beakers at the end of the test medium renewal period.

b) Samples without food and test animals incubated during the renewal periods under the test conditions were also taken.

All samples were stored deep-frozen (at about -20 °C) immediately after sampling. Based on a pre-experiment (non-GLP) for investigation of the storage stability, neo-decanoic acid proved to be stable in the test water under these storage conditions.
Fresh test media samples and stability samples were analyzed. Since in a pre-experiment (non-GLP) the presence of food in the stability samples proved not to influence the stability of the neo-decanoic acid, stability samples containing food were analyzed exclusively.

Test solutions

Vehicle:

no

Details on test solutions:

Prior to the start of the test and prior to each test medium renewal, individual dispersions of the test item were prepared:

Loading Rate (mg/L) Preparation
3.2 6.39 to 6.44 mg test item in 2000 mL test water
10 20.00 to 20.20 mg test item in 2000 mL test water
32 63.99 to 64.32 mg test item in 2000 mL test water
100 200.00 to 200.63 mg test item in 2000 mL test water


The dispersions were stirred for 23 hours at room temperature in the dark to dissolve a maximum amount of the different test item components in the test water. This long stirring period was chosen based on the low solubility of the test item in the test medium. In a pre-experiment (non-GLP) a prolongation of the stirring time to 96 hours did not significantly increase the amount of test item (dissolved organic carbon) in the 100 mg/L-WAF. After 23 hours stirring, the content of DOC in the test medium was 14.77 and 14.64 mg/L. After 96 hours, 14.69 and 14.82 mg DOC/L were measured. Thus, the stirring period of 23 hours was considered appropriate.

After stirring, the dispersions were left to settle for 1 hour to separate undissolved test material from the test water and the middle layers were drawn off with a Teflon tube and tested as WAFs.

Due to technical reasons, the WAF with the lowest loading rate of 1.0 mg/L was prepared as dilution of the WAF with the loading rate of 3.2 mg/L.

The test media were freshly prepared prior to test start and prior to each test medium renewal.

The preparation of the test media was based on the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

The study was performed with females of the species Daphnia magna Straus. A clone of this species (defined by the supplier as clone 5) was originally supplied by the University of Sheffield/UK in 1992. Since this date, the clone is successfully bred at Harlan Laboratories in culture medium identical to the medium used for the test (see Section 3.4). The temperature and light conditions were identical to those of the test.

During breeding, daphnids are generally fed three times a week with an algal suspension of the green algae Scenedesmus subspicatus CHODAT, Strain No. 86.81 SAG, supplied by the Collection of Algal Cultures (SAG, Institute for Plant Physiology, University of Göttingen, 37073 Göttingen / Germany) and cultivated at Harlan Laboratories under standardized conditions or a mixture of this algal suspension and a commercial fish diet (Tetra Min Hauptfutter, supplied by TETRA-Werke, 49304 Melle / Germany).

Each stock animal was maintained separately in a 100 mL glass beaker filled with about 80 mL culture medium and was transferred twice a week to fresh medium. The condition of the stock animals was frequently checked. No signs of stress were observed and the brood stock was healthy.

The daphnids used for the test originated from parental daphnids that were at least 14 days old but not older than four weeks and were not first brood progeny. At the start of the test, the test animals were less than 24 hours old.

The test method and test species, Daphnia magna, are recommended by the test guidelines.

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Post exposure observation period:

not applicable

Test conditions

Hardness:

2.5 mmol/L (= 250 mg/L as CaCO3)

Test temperature:

The water temperature was 20-21 °C during the test period.

pH:

During the test period, the pH of the test media and control ranged from 7.5 to 8.1 (see attached Table 6).

Dissolved oxygen:

The dissolved oxygen concentrations were at least 7.0 mg/L (see attached Table 7).

Salinity:

according to OECD test guideline

Nominal and measured concentrations:

In order to assess the toxicity of the sparingly soluble multi-component test item E0286P/040A to Daphnia magna, water accommodated fractions (WAFs) with the loading rates of 1.0, 3.2, 10, 32, and 100 mg/L were tested. Additionally, a control group was tested in parallel.
At the beginning of the test medium renewal periods, the concentrations of the test item component neo-decanoic acid varied between 3.15 and 11.5 mg/L in the test media of the loading rate of 32 mg/L and between 15.9 and 23.7 mg/L in the test media of the loading rate of 100 mg/L. Over the renewal periods of 48 and 72 hours the concentrations of neo-decanoic acid were stable in the test media. In the control, all concentrations were below the Limit of Quantification of 0.0787 mg neo-decanoic acid/L.

Details on test conditions:

The test was conducted in reconstituted water (Elendt M7 medium). Before use, the test water was aerated until oxygen saturation. During the test, the test media were not aerated.
For further information on test water please see section "Any other information on materials and methods incl. tables" below.

The test was performed in 100 mL glass beakers containing 80 mL of test medium. The test vessels were covered with glass plates to reduce the loss of water by evaporation and to avoid the entry of dust into the solutions. The test vessels were labeled with the study number and all necessary additional information to ensure unique identification.

This semi-static test with test medium renewals every 48 or 72 hours was performed in a temperature-controlled room with continuous monitoring of the room temperature. The water temperature was maintained at 20 21 °C.

A 16-hour light to 8-hour dark cycle with a 30 minute transition period was used. Light intensity during the light period was between approximately 485 and 640 Lux. At test medium renewal, the surviving test animals were carefully transferred by means of glass tubes from the old test vessels into the freshly prepared test medium.

The test animals were fed daily (with the exception of Day 4 (Sunday)) with a food mixture containing a suspension of green algae of the species Desmodesmus subspicatus (freshly grown at Harlan Laboratories) and a fish food suspension.

The fish food suspension was prepared by dispersing 10 g of powdered commercial fish diet (TETRA MIN Hauptfutter, obtained from TETRA-Werke, 49304 Melle / Germany) in 500 mL of test water. The suspension was allowed to stand for 4 hours. Then, 400 mL of the supernatant were taken, diluted 1:1 with test water and boiled. The suspension was stored deep frozen in small quantities until use.

The carbon contents of the algal and fish food suspensions were determined using a Shimadzu TOC 5000A Analyzer. The food amount (based on the measured concentrations of the total organic carbon (TOC) in the food suspensions) was 0.20 mg TOC per Daphnia and day.

The following loading rates of E0286P/040A were tested: 1.0, 3.2, 10, 32, and 100 mg/L. Additionally, a control (test water without test item) was tested in parallel. The selection of the test concentrations was based on the results of a range-finding test (non-GLP).

The test was performed semi-statically. The test media of all treatments were renewed on Days 2, 5, 7, 9, 12, 14, 16, and 19 (every Monday, Wednesday, and Friday). The study was started with 10 daphnids per treatment. Each test animal was kept individually in a glass beaker. The test animals were randomly distributed to the test vessels. The test duration was 21 days.

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

21 d

Dose descriptor:

EL50

Effect conc.:

94 mg/L

Conc. based on:

test mat.

Remarks:

loading rate

Basis for effect:

reproduction

Remarks on result:

other: 95% confidence interval: 93 - 94 mg/L

Effect concentrations 2

Duration:

21 d

Dose descriptor:

NOELR

Effect conc.:

32 mg/L

Conc. based on:

test mat.

Remarks:

loading rate

Basis for effect:

reproduction

Effect concentrations 3

Duration:

21 d

Dose descriptor:

LOELR

Effect conc.:

100 mg/L

Conc. based on:

test mat.

Remarks:

loading rate

Basis for effect:

reproduction

Details on results:

Analytical Results
Concentrations of Neo-decanoic Acid in the Test Media
At the beginning of the test medium renewal periods, the concentrations of the test item component neo-decanoic acid varied between 3.15 and 11.5 mg/L in the test media of the loading rate of 32 mg/L and between 15.9 and 23.7 mg/L in the test media of the loading rate of 100 mg/L . Over the renewal periods of 48 and 72 hours the concentrations of neo-decanoic acid were stable in the test media. In the control, all concentrations were below the Limit of Quantification of 0.0787 mg neo-decanoic acid/L.

Total Organic Carbon Content of the Test Media
At the beginning of the test medium renewal periods, the TOC-content of the test media varied between 3.1 and 9.4 mg/L at the loading rate of 32 mg/L and between 14.0 and 20.5 mg/L at the loading rate of 100 mg/L. At the end of the renewal periods of 48 and 72 hours, the TOC-content varied between 2.7 and 9.4 mg/L at the loading rate of 32 mg/L and between 13.2 and 20.6 mg/L at the loading rate of 100 mg/L. In the control, the TOC-values were below the LOQ of 1 mg/L. Thus, the presence of the test item in the test item treated test media could be verified. Furthermore, the measured TOC-contents of the test media followed the same pattern as the analytical determination of neo-decanoic acid.

The analyzed carbon contents of the standard samples used for the calibration of the system deviated by at maximum 3% from the nominal value of 50 mg C/L and, thus, the measurements were considered to be valid.


Biological Results
In the control and up to and including the loading rate of 32 mg/L, the survival of the test animals at the end of the test was at least 90% . At the highest loading rate of 100 mg/L, the survival was 80%. A mortality of up to 20% is regarded as natural and tolerated by the test guideline. However, at the highest loading rate tested, the mortality was interpreted as a slight toxic effect caused by the test item also refering to the effects on mean reproduction described below. Thus, the survival of Daphnia magna after 21 days was only slightly reduced at the loading rate of 100 mg/L.

The first young offspring released from their parent animals were recorded in all treatments at observation Day 9. Thus, the time of the first brood was not affected by the test item up to and including the loading rate of 100 mg/L.

The mean reproduction rate of the daphnids in the control was 143 ± 12 living offspring per adult (mean ± standard deviation). No significant inhibitory effect of the test item on the mean reproduction rate was determined up to and including the loading rate of 32 mg/L (Welch t-test, one-sided smaller, alpha = 0.05). At the highest loading rate of 100 mg/L, the mean reproduction rate of surviving daphnids was statistically significantly reduced to on average 68 daphnids (48% compared to the control).

With the exception of the reported mortality and reduced reproduction rates, visible abnormalities were only observed at one control test animal recorded dead on Day 19. Already on Day 9 the daphnid appeared pale. No other visible abnormalities were observed in the test animals during the test.

Conclusion
Taking into account the effects on survival and reproduction of the test animals, the highest loading rate of E0286P/040A tested without toxic effects after the exposure period of 21 days (21 day NOELR) was 32 mg/L. The lowest loading rate tested with toxic effects (21 day LOELR) was determined to be 100 mg/L due to the mortality rate and statistically significantly reduced mean reproduction rate of Daphnia magna at this treatment.

General Results
During the test period, the pH of the test media and control ranged from 7.5 to 8.1. The dissolved oxygen concentrations were at least 7.0 mg/L. The water temperature was 20-21 °C during the test period.

No remarkable observations were made concerning the appearance of the test media. All test media were clear solutions throughout the test medium renewal periods.

Results with reference substance (positive control):

not applicable

Reported statistics and error estimates:

The mean reproduction rates of the daphnids at the test concentrations were compared to the control by multiple Welch t-tests. Additionally, the EL50 for the inhibition of the reproduction rate after 21 days was calculated by Probit Analysis using weighted linear regression.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

loading rate of E0286P/040A tested without toxic effects after the exposure period of 21 days (21 day NOELR) was 32 mg/L.

The lowest loading rate tested with toxic effects (21 day LOELR) was determined to be 100 mg/L due to the mortality rate and statistically significantly reduced mean reproduction rate of Daphnia magna at this treatment.

Executive summary:

The effect of the test item E0286P/040A on the survival and reproduction of Daphnia magna was investigated in a semi-static test over 21 days following the OECD Guidelines for Testing of Chemicals, No. 211 (2008): “Daphnia magna Reproduction Test” and the EU Commission Directive 92/69/, C.20: “Daphnia magna Reproduction Test".

 

In order to assess the toxicity of thesparinglysoluble multi-component test item E0286P/040A to Daphnia magna, water accommodated fractions (WAFs) with the loading rates of 1.0, 3.2, 10, 32, and 100 mg/L were tested. Additionally, a control group was tested in parallel.

 

For preparation of the WAFs, individual dispersions of the test item with the loading rates as mentioned above were prepared. The dispersions were stirred for 23 hours to dissolve a maximum amount of the different components of the test item in the dispersion.After stirring, each dispersion was left to settle for 1 hour and the middle layer of the water column was drawn off using a Teflon tube to be tested as WAF.

The preparation of the test media was based on the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures (2000).

 

At the beginning of the test medium renewal periods, the concentrations of the test item component neo-decanoic acid varied between 3.15 and 11.5 mg/L in the test media of the loading rate of 32 mg/L and between 15.9 and 23.7 mg/L in the test media of the loading rate of 100 mg/L. Over the renewal periods of 48 and 72 hours the concentrations of neo-decanoic acid were stable in the test media. In the control, all concentrations were below the Limit of Quantification of 0.0787 mg neo-decanoic acid/L.

 

The measured-contents of the test media followed the same pattern as the analytical determination of the neo-decanoic acid. At the beginning of the test medium renewal periods, the-content of the test media varied between 3.1 and 9.4 mg/L at the loading rate of 32 mg/L and between 14.0 and 20.5 mg/L at the loading rate of 100 mg/L. At the end of the renewal periods of 48 and 72 hours, the-content varied between 2.7 and 9.4 mg/L at the loading rate of 32 mg/L and between 13.2 and 20.6 mg/L at the loading rate of 100 mg/L. In the control, the-values were below the LOQ of 1 mg/L.

 

Thus, the presence of the test item in the test item treated test media of 32 and 100 mg/L could be verified.

 

The results of the study can be summarized as follows:

 

	Loading rate of the test item (mg/L)
	Control	1.0	3.2	10	32	100
Mortality (%) after 21 days of exposure	10	0	0	0	0	20
Mean reproduction rate (living offspring per surviving adult)	142.8	143.6	145.8	138.9	151.7	68.1*
Mean reproduction rate in % of control	100.0	100.6	102.1	97.3	106.2	47.7

 

*    statistically significantly lower than the control value
(results of Welch t-test, one-sided smaller,a= 0.05)

 

 

Parameter	Inhibition of reproduction rate
	(21 days)
NOELR (mg/L)	32
LOELR (mg/L)	100
EL50  (mg/L)	94
95% confidence interval	93 - 94