4-Piperidinone, 2,6-diethyl-2,3,6-trimethyl-1-(1-phenylethoxy)-

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Performed according to OECD-Guideline under GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Details on test material:

Identity: TKA 40270 (CGPS 345)
Batch no.: Lot 02243FC7, Op. 1/07
Purity: Confidential information (see Analytical Certificate No.38291-1)
Expiration date: 01-Oct-2012
Solubility in water: <0.21 mg/L
Aggregate state / physical form at room temperature: Liquid
Color: Colorless to yellow
Storage conditions: Storage at under 18 °C may lead to partial solidification. To reliquify, heat contents to 25-35 °C. Protect from light. (At RCC: At room temperature at about 20 °C, away
from direct sunlight.)

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

For measurement of the actual concentrations of the test item, duplicate samples were taken from
the test media of all test concentrations at the start of the test (without algae) and at the end of the
test (containing algae). At the same sampling times, duplicate samples were also taken from the
control. For the 72-hour stability samples, the contents of the test flasks of each treatment were
combined.
In addition, a vessel containing the undiluted filtrate was incubated without algae under test
conditions.
The samples were analyzed immediately after sampling. In order to stabilize the samples, 1 mL
acetonitrile per mL sample was added.

Test solutions

Vehicle:

no

Details on test solutions:

Due to the low water solubility of the test item, a dispersion with the loading rate of 100 mg/L was prepared by dispersing 150.5 mg of the test item
in 1500 mL of test water using intense stirring. No auxiliary solvent or emulsifier was used. The dispersion was stirred by a magnetic stirrer at room temperature in the dark over 96 hours to dissolve a maximum amount of the test item in the dispersion.
The stirring period of 96 hours was selected according to the results of a pre-test (non-GLP) which showed that the concentration of dissolved test
item was increasing over the stirring period of 96 hours.
After the 96-hour stirring period, the dispersion of the test item was filtered through a membrane filter (Schleicher & Schuell, Type NC45, pore size 0.45 µm). The undiluted filtrate was used as the highest concentrated test medium and as stock solution for the preparation of the test media of lower test concentrations. For the preparation of the test media of the lower test concentrations, the filtrate was diluted with test water. The test media were prepared just before the start of the test (= addition of algae).

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

The test organism used for the study was 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). The algae were cultivated in RCCs laboratories under standardized conditions according to the test guidelines.
Due to the division of the genus Scenedesmus into the genera Scenedesmus and Desmodesmus, Scenedesmus subspicatus was transferred to the new genus Desmodesmus [2, 3]. Thus, Scenedesmus subspicatus was renamed as Desmodesmus subspicatus. However, the former name Scenedesmus subspicatus as specified in the guidelines is used in this report.
An inoculum culture was set up four days before the start of the test. The algae were cultivated under the test conditions. The inoculum culture was diluted threefold one day before the start of the test to ensure that the algae were in the exponential growth phase when used to inoculate the test solutions.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

Exponentially growing cultures of this algal species were exposed to the test item over a period of 72 hours and
the inhibition of algal growth in relation to control cultures were assessed over several
generations.

Test conditions

Hardness:

24 mg/L as CaCO3

Test temperature:

22-23°C

pH:

The pH values was between 8.2 and 8.6 at the end of the test.

Nominal and measured concentrations:

Nomerial Concentration: 100 mg/L (loading rate) and dilutions of the filtrate 1:2.2, 1:4.6, 1:10, 1:22
Measured concentration: Undiluted filtrate: 0.35 mg/L; Dilution 1:2.2: 0.15 mg/L ; Dilution 1:4.6:

Details on test conditions:

The test flasks were incubated in a temperature-controlled water bath at a temperature of 22-23 °C and illuminated by fluorescent tubes (Philips TLD 36W/840), installed above the test flasks. The mean measured light intensity at the level of the test solutions was approximately 5000 Lux (range:
4500 to 5400 Lux, measured at nine places in the experimental area).

Reference substance (positive control):

yes

Remarks:

For evaluation of the algal quality and the experimental conditions, potassium dichromate is tested as a positive control twice a year to demonstrate satisfactory conditions of the test.

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 4

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Effect concentrations 5

Duration:

72 h

Dose descriptor:

EC0

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Effect concentrations 6

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Details on results:

The test item had no significant inhibitory effect on the growth of the algae (growth rate and yield) during the test period of 72 hours up to and
including the highest test concentration of 100 mg/L (loading rate ) corresponding to a measured concentrtion of the test item in the filtrate of 0.35 mg/L (results of Dunnetts tests, one-sided, a = 0.05).
The test concentration of 100 mg/L (loading rate) corresponding to a measured concentration of the test item in the fil.trate of 0.35 mg/L, thereforewas determined to be the 72-hour NOEC. The 72-hour LOEC was higher than 100 mg/l (loading rate). o.35 mg/L was representing the maximum
concentration of the test item, which could be dissolved in the test water.

Results with reference substance (positive control):

The latest result of the positive control test in 2007 (72-h ECSO for the growth rate: 0.64 mg/L, RCC
Study No. B673S3) showed that the toxic performance was valid and within the historical range
ofthe RCC laboratory (from 1996 to 2007: 72-h ECSO: 0.44-1.16 mg/L).

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The test item TKA 40270 (CGPS 345) had no toxic effects on the algae up to its water solubility limit in test water under the present conditions of the test (corresponding to a loading rate of 100 mg/L).

Executive summary:

The test item had no significant inhibitory effect on the growth of the algae (growth rate and yield) during the test period of 72 hours up to and including the highest test concentration of 100 mg/L (loading rate ) corresponding to a measured concentrtion of the test item in the filtrate of 0.35 mg/L (results of Dunnetts tests, one-sided, a = 0.05).

The test concentration of 100 mg/L (loading rate) corresponding to a measured concentration of the test item in the fil.trate of 0.35 mg/L, thereforewas determined to be the 72-hour NOEC. The 72-hour LOEC was higher than 100 mg/l (loading rate). o.35 mg/L was representing the maximum concentration of the test item, which could be dissolved in the test water.

The test item were analytically determined concentrations of the test item in the test media (dilution 1:2.2 and the undiluted filtrate) were 0.15 and 0.35 mg/L, respectively. During the test period of 72 hours, a decrease of test item concentration was determined in the presence of algae. At the end of the test, a concentration of 0.19 mg/L was determined in the undiluted filtrate with the loading rate of 100 mg/L. However, in samples from the undiluted filtrate incubated under the test conditions for 72 hours without algae, the test item was shown to be stable (measured concentration of 0.31 mg/L). Thus, the reason for the decrease of the test item in the samples containing algae was adsorption to algal cells. Consequently, the test item was not lost from the test system and the biological results are related to the initially measured test concentrations, as well as to the loading rate.