2-benzyl-2-dimethylamino-4'-morpholinobutyrophenone

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:

2009-03-16, 2009-04-08

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Deviations:

yes

Remarks:

Temperatures (18.9 to 21.0°C) slightly exceeded the range of ± 1°C described in the guideline by 0.1°C.

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD Guideline 23: Aquatic Toxicity Testing of Difficult Substances and Mixtures

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 10 mg/L
- Sampling method: Samples of fresh test solutions were taken on days 0, 7, and 14 from the beakers used to prepare the solutions. Samples of aged test solutions were taken on days 2, 9, and 16 after the content of the replicates of a given concentration was mixed. A volume of 25 mL methanol was added into the samples. All samples were shaken and sonicated for 10 minutes.
- Sample storage conditions before analysis: Frozen and protected from daylight, in good condition

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: supersaturated stock solution (dispersion) in Medium M4. The test item was mixed into the test water as homogeneously as possible using ultrasonication for approximately 5 minutes followed by intense stirring on a magnetic stirrer at ambient temperature in the dark for two days to dissolve a maximum amount of the test item. After that, and immediately before use in the test, an appropriate volume of the dispersion was filtered (pore size: 0.45 µm) using a glass fibre filter. The filtrate was used as the test solution. The test solutions were handled protected from daylight or UV-light.
- Evidence of undissolved material: None

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water flea
- Strain/clone: Clone 5
- Source: ECT Oekotoxikologie GmbH since October 11, 2000
- Age of parental stock: 19 - 23 d (offspring used was not the first brood of these parental daphnids)
- Feeding during test
- Food type: fresh algae (Desmodesmus subspicatus) suspension
- Amount: 0.1 to 0.2 mg C/daphnia/d
- Frequency: three times per week

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES: One day before starting the test all young daphnids were removed from the selected holding vessels which contained adult daphnids. At least 6 hours before placing young daphnids into the test vessels the newborn daphnids were separated from parental daphnids which were kept in the selected holding vessels.

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

21 d

Hardness:

250.0 mg/L CaCO3

Test temperature:

18.9 to 21.0°C (manual measurement; n = 12); 18.8 to 20.4°C (online measurement; n = 1003)

pH:

7.6 - 7.8

Dissolved oxygen:

94 - 95 % (8.4 - 8.6 mg/L)

Nominal and measured concentrations:

10 mg/L (nominal) 0.0596 - 0.2768 mg a.i./L (measured; a.i. = active ingredient) (table 4)

Details on test conditions:

TEST SYSTEM
- Test vessel: Beakers, covered by glass lids
- Type (delete if not applicable): open
- Material, nominal volume, fill volume: Glass, 100 mL, 50 - 100 mL (fill volume)
- Aeration: None
- Renewal rate of test solution: 3 times per week
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 12
- No. of vessels per control (replicates): 12

TEST MEDIUM: Elendt medium M4
- Alkalinity: 40- 60 mg/L CaCO3
- Conductivity: 615 - 617 µS/cm
- Culture medium different from test medium: No
- Intervals of water quality measurement: once per test week

OTHER TEST CONDITIONS
- Adjustment of pH: None
- Photoperiod: 16 h light / 8 h dark
- Light intensity: 0.09 to 0.18 µE me-2 se-1 (n = 5)

EFFECT PARAMETERS MEASURED: fecundity (live, aborted eggs, and dead offspring) (daily from day 7); immobility (3 times per week); mortality (daily from day 7) and length of surviving parental daphnids (at the end of the test)

Reference substance (positive control):

yes

Remarks:

Potassium dichromate (K2Cr2O7)

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

>= 0.21 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

> 0.21 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

>= 0.21 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

immobilisation

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

> 0.21 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

immobilisation

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

>= 0.21 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

morphology

Remarks:

length of parental daphnids

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

> 0.21 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

morphology

Remarks:

length of parental daphnids

Details on results:

- Mortality of parent animals: 1
- No. of offspring produced per day per female: 8 - 57
- Body length and weight of parent animals: 4.17 - 4.57 mm
- Type and number of morphological abnormalities: None
- Type and number of behavioural abnormalities: None
- Time to first brood release or time to hatch: Day 8
- Other biological observations: Daphnia sticking to the water surface; Daphnia showing reduced swimming activity; aborted eggs; dead juvenile daphnids.
- Effect concentrations exceeding solubility of substance in test medium: None

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Relevant effect levels: EC50(24 h) of 1.76 mg/L

Reported statistics and error estimates:

Cumulative number of living offspring per parent animal alive at the end of the test and average length of individual parental daphnids and reduction of length in percentage compared to the controls after 21 d were analyzed statistically (table 1 and 2 resp.).

Table 1: Cumulative number of living offspring per parent animal alive at the end of the test.

concentration level	nominal concentration [% of WAF at 10 mg/L]	mean number of offspring	sd	cv [%]	n
C0	Control	132.8	15.82	11.9	12
C1	100	128.3	19.39	15.1	11

sd = standard deviation; cv = coefficient of variation; n = number of parent animals alive at the end of the test.

Table 2: Average length of individual parental daphnids and reduction of length in percentage compared to the controls after 21 d.

concentration level	nominal concentration [% of WAF at 10 mg/L]	minimum [mm]	maximum [mm]	mean value	standard deviation	n
C0	Control	4.17	4.47	4.40	0.085	12
C1	100	4.11	4.57	4.37	0.136	11

n: number of parent animals alive at the end of the test.

Table 3: Cumulative number of offspring per surviving parental daphnid at the end of the test.

Replicate	Control	100 % of WAF at 10 mg/L]
A	116	104
B	144	138
C	147	117
D	125	116
E	129	147
F	131	123
G	165	130
H	149	94
I	131	140
J	118	145
K	110	-
L	128	157

Table 4: Summary of measured concentrations in fresh and aged test solutions throughout the test.

nominal concentration (mg/L)	test period (days)	age of solution (days)	measured concentration (mg/L)	mean measured concentration (mg/L)
Control	0	0	< 30% LOQ
	2	2	< 30% LOQ
	9	2	ND
	16	2	ND
WAF at 10 mg/L	0	0	0.0596
	2	2	0.2064
	7	0	0.2623
	9	2	0.2169
	14	0	0.2768
	16	2	0.2568	0.2131

WAF = water-accommodated fraction

Validity criteria fulfilled:

yes

Conclusions:

No chronic effects within the range of solubility. The 21-day NOEC of the test material to Daphnia magna was determined to be >/= 0.21 mg/l (measured) (above the limit of solubility).

Executive summary:

In this guideline (OECD 211) study conducted to GLP standards, the long-term (21 day) NOEC of the test material (EC 404-360-3) to Daphnia magna was determined to be >/= 0.21 mg/l (measured). The test was conducted using a Water Accomodated Fraction with a loading rate of 10 mg/l. No chronic effects were observed over the duration of the test. The study result does not trigger classification of the test material as hazardous to the aquatic environment under the criteria of the EU Classification, Labelling, and Packaging (CLP) regulation, as no toxic effects were observed up to the limit of solubility.

Description of key information

Study conducted to recognised training guidelines with GLP

Additional information

A semi-static GLP guideline study according to OECD 211 has been conducted (ECT Oekotoxikologie GmbH 2009). Due to the low solubility of the test item within the test medium a saturated solution has been used. Under the conditions of the test, the test item showed no effect on the test organisms at 100% of the saturated solution at 10 mg/L. The mean measured concentration was determined to be 0.21 mg/L. The NOEC and LOEC values of the parameter fecundity (cumulative number of living offspring per parent animal alive at the end of the test), immobility and length of parental daphnids are all above the maximum solubility.

Therefore, the test substance is with high probability chronically not harmful to aquatic organisms.