Adenine

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-09-18 to 2018-09-20

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

2004

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Sampling method: At the start of the exposure and at renewal (0 and 24 hours), samples of the freshly prepared test item concentrations were taken and analyzed. At renewal and at the end of the exposure (24 and 48 hours), samples of the 24-hours old media were taken directly from the test vessels and analyzed.
- Sample storage conditions before analysis: No

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A stock solution with a nominal concentration of 100 mg/L of the test item was prepared with dilution water directly prior to the start of the exposure (at day 0) and at the day of the renewal of the test solutions (at day 1). An appropriate amount of the test item was weighed in and transferred to a glass flask with an appropriate amount of dilution water. This stock solution was prepared by ultrasound treatment for 30 minutes at room temperature. Care was taken to avoid bacterial contamination. 5 test item concentrations in a geometric series with a separation factor of 2 (6.25 - 12.5 - 25.0 - 50.0 - 100 mg/L) were prepared.
- Controls:
Blank control: Dilution water without test item incubated under the same conditions as the test groups
Refernce control: A reference test with potassium dichromate was conducted as an acute immobilization test in Elendt M4 medium under static conditions with a test duration of 24 hours once per month in order to prove the validity of the test system and test conditions at the test facility.
- Evidence of undissolved material: No

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water flea
- Strain/clone: Clone 5
- Source: Institut fur Wasser-, Boden- und Lufthygiene (WaBoLu), 14195 Berlin, Germany
- Age of test animals: Less than 24 hours old
- Feeding during test: No

ACCLIMATION
- Acclimation period: Not necessary (culturing conditions same as during test)

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES: Less than 24 hours old daphnids from a healthy stock were used for the study. Juvenile daphnids were removed from the culture vessels at the latest 24 hours before the start of the exposure and discarded. The juveniles bom within the following period of max. 24 hours preceding the exposure were used for the test. No first brood progeny was used for the test.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

No

Hardness:

265 mg CaCO3/L

Test temperature:

0 hours: 20.1 °C
24 hours: 20.4 °C

pH:

0 hours: 7.46
24 hours: 7,45

Dissolved oxygen:

o hours: 9.00 mg/L
24 hours: 8.95 mg/L

Salinity:

Not applicable

Conductivity:

0 hours: 592 µS/cm
24 hours: 591 µS/cm

Nominal and measured concentrations:

Nominal: 6.25, 12.5, 25.0, 50.0 and 100 mg/L.
Mean measured: 6.46,12.2, 24.0, 49.1 and 97.8 mg/L (old media, 24 hours); 6.11, 12.1, 24.0, 49.3 and 98.4 mg/L (old media, 48 hours)

Details on test conditions:

TEST SYSTEM
- Test vessel: Beakers
- Material, size, headspace, fill volume: Glass, 4 (ID) x 7 (H) cm, 50 mL capacity, loosely covered with watch glasses, 20 mL fill volume
- Aeration: No
- Renewal rate of test solution: 24 hours
- No. of organisms per vessel: 5
- No. of vessels per concentration: 4
- No. of vessels per control: 4

TEST MEDIUM
- Preparation of dilution water: Elendt M4 using ultra-pure water, Milli-Q system

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16/8 hours light/dark cycle
- Light intensity: Diffuse light, light intensity of max. 1500 Iux

EFFECT PARAMETERS MEASURED:
Immobilisation

RANGE-FINDING STUDY
- Test concentrations (nominal): 100, 10.0 and 1.00 mg/L
- Results used to determine the conditions for the definitive study: Yes

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

24 h

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC100

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

24 h

Dose descriptor:

EC100

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

- Observations on body length and weight: No
- Other biological observations: No
- Mortality of control: No
- Other adverse effects control: No
- Abnormal responses: No
- Any observations that might cause a difference between measured and nominal values: No, all tested concentration levels were visually clear and colorless throughout the exposure.
- Effect concentrations exceeding solubility of substance in test medium: No

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Relevant effect levels: EC50
- Limit test: No
- Dose-response test: Yes
- ECx: 24-h EC50 = 1.89 mg/L

Reported statistics and error estimates:

The EC10, 50, 100-values (after 24 and 48 hours) were empirically derived from the observation data.

Table 1: Immobilization Rates after 24 and 48 hours of Exposure in the Definitive Test (n = 20, divided into 4 replicates with 5 daphnids each)

Nominal	IMMOBILIZATION [%]
test item	24 hours					48 hours
concentration	Replicates					Replicates
[mg/L]	1	2	3	4	MV	1	2	3	4	MV
100	0	0	0	0	0	0	0	0	0	0
50	0	0	0	20	5	0	0	0	20	5
25	0	0	0	0	0	0	0	0	0	0
12.5	0	0	0	0	0	0	0	0	0	0
6.25	0	0	0	0	0	0	0	0	0	0
Control	0	0	0	0	0	0	0	0	0	0

Table 2 Absolute Numbers of immobile Daphnids after 24 and 48 h of Exposure in the Definitive Test (n = 20, divided into 4 replicates with 5 daphnids each)

Nominal test item concentration [mg/L]	NUMBER OF IMMOBILE DAPHNIDS / TOTAL NUMBER OF DAPHNIDS
	24 hours					48 hours
	Replicates					Replicates
	1	2	3	4	Sum	1	2	3	4	Sum
100	0/5	0/5	0/5	0/5	0/20	0/5	0/5	0/5	0/5	0/20
50	0/5	0/5	0/5	0/5	0/20	0/5	0/5	0/5	0/5	0/20
25	0/5	0/5	0/5	0/5	0/20	0/5	0/5	0/5	0/5	0/20
12.5	0/5	0/5	0/5	0/5	0/20	0/5	0/5	0/5	0/5	0/20
6.25	0/5	0/5	0/5	0/5	0/20	0/5	0/5	0/5	0/5	0/20
Control	0/5	0/5	0/5	0/5	0/20	0/5	0/5	0/5	0/5	0/20

Table 3: Measured Concentrations and Percent of Nominal Concentration of the Test Item during the Definitive Test

Sampling date	Fresh media, 0 hours		Old media, 24 hours		Fresh media, 24 hours		Old media, 48 hours
Nominal concentration [mg/L]	Meas. conc. [mg/L]	%	cone.	%	cone.	%	cone.	%
100	97.6	98	97.8	98	98.6	99	98.4	98
50	48.8	98	49.1	98	49.3	99	49.3	99
25	23.9	96	24	96	24.1	96	24	96
12.5	11.9	95	12.2	98	12.7	102	12.1	97
6.25	6.07	97	6.46	103	6.46	103	6.11	98
Control	< LOQ		< LOQ		< LOQ		< LOQ

Meas. conc. = measured concentration of the test item, mean value of 2 injections, dilution factors taken into account

% = percent of the nominal concentration of the test item

LOQ = limit of quantification of the analytical method (1.80 mg/L of the test item)

Validity criteria fulfilled:

yes

Conclusions:

In a semi-static acute immobilization test with Daphnia magna according to OECD TG 202, the 48-hour EC50 was determined to be > 100 mg/L (nominal).

Executive summary:

An acute immobilization test to Daphnia magna according to OECD TG 202 and GLP principles was carried out to determine the EC10-, EC50- and EC100-values of the test item after 24 and 48 hours of exposure under semi-static conditions (renewal of test solutions after 24 hours), using the following nominal test item concentrations: 6.25, 12.5, 25.0, 50.0 and 100 mg/L. 20 daphnids, divided into 4 replicates, each with 5 daphnids, were used for each concentration level and the control (dilution water without test item incubated under the same conditions as the test groups). The suitability of the test system was confirmed by a separate acute immobilisation test with the reference item potassium dichromate. The test item concentrations in the test solutions were verified by HPLC-DAD analysis. All effect concentrations (EC10/50/100) given are based on the nominal concentrations, since the measured test item concentrations were within the range of ± 20% of the nominal concentrations. In result, the 24-hour and 48-hour EC10, EC50 and EC100 were determined to be > 100 mg/L. All validity criteria of the test guideline were fulfilled.

Description of key information

In a semi-static acute immobilization test with Daphnia magna according to OECD TG 202, the 48-hour EC50 was determined to be > 100 mg/L (nominal) (reference 6 .1.3-1).

Additional information

An acute immobilization test to Daphnia magna according to OECD TG 202 and GLP principles was carried out to determine the EC10-, EC50- and EC100-values of the test item after 24 and 48 hours of exposure under semi-static conditions (renewal of test solutions after 24 hours), using the following nominal test item concentrations: 6.25, 12.5, 25.0, 50.0 and 100 mg/L. 20 daphnids, divided into 4 replicates, each with 5 daphnids, were used for each concentration level and the control (dilution water without test item incubated under the same conditions as the test groups). The suitability of the test system was confirmed by a separate acute immobilisation test with the reference item potassium dichromate (24-hour EC50 = 1.89 mg/L). The test item concentrations in the test solutions were verified by HPLC-DAD analysis. All effect concentrations (EC10/50/100) given are based on the nominal concentrations, since the measured test item concentrations were within the range of ± 20% of the nominal concentrations. In result, the 24-hour and 48-hour EC10, EC50 and EC100 were determined to be > 100 mg/L. All validity criteria of the test guideline were fulfilled.