Calcium acetylide

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:

1999

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

The test samples of the acute Daphnia immobilisation test were received in Sarstedt tubes.
The test samples were stored at -18 °C until sample analysis.
Each sample got 1 mg potassium/100 ml in according to the calibration samples.

Vehicle:

no

Details on test solutions:

1) Description of the method
Daphnia, 2 up to 24 hours old, were kept in samples of defined water (ISO 6341) to which different concentrations of the test substance were added.
The aim was to cause toxic effects within 24 hours as well as 48 hours and to determine the concentrations that cause effects ranging from no observable effect up to 100 % immobilization of Daphnia.
To determine the dosage for the main-test, a range-finding test was performed. Before the beginning and at the end of the test the pH, the content of dissolved oxygen and the temperature were measured in the test solutions.

2) Preparation of the test solutions
The test substance Calciumcarbide is soluble in water. Therefore, the test solution was prepared with test water by weighing and transferring the test substance directly in the test water. The test solutions were prepared corresponding to the required test concentrations (geometric series with factor 1.5) in the following way:
- Filling the flasks with 2000 ml test water
- Transfer of the Daphnia into the test vessel with the test water
- Addition of the test substance weighed per vessel directly into the test water with the Daphnia
The control vessels contained dilution water only.
For the beginning of the test the dilution water was prepared the day before and aerated overnight in the same cabinet and at the same temperatures as the Daphnia test was performed.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Source: Landesamt für Umweltschutz, Karlsruhe, Germany
- Origin of the animals used in the test: holding and breeding in the testing facility under standardized laboratory conditions; originated from the same culture batch
- Holding conditions
* Breeding medium: M 4 medium
* Preparation: with air saturated deionized water (conductivity £ 1.5 mS/cm) according to ELENDT (1990);the sum of calcium and magnesium ions in the M 4 medium was 2.7 ± 0.5 mmol/l; the proportion of Ca to Mg ions was 4:1 and that of Na to K ions was 10:1; the total alkalinity of this solution amounted 0.9 ± 0.1 mmol/l; before use the air saturated M 4 medium was stored for at least 12 h
* Breeding: breeding was performed in glass beakers (50 ml) with M 4 medium (50 ml/Daphnia); Daphnia were transferred to new M 4 medium twice weekly;
animals were kept at the same temperature and light conditions as in the test itself; test animals were separated from the adults by means of a pipette
* Food: alga Scenedesmus subspicatus
* Feeding: daily; Daphnia of different age were fed with a defined suspension of algae: 10 days old: 1.5 ml/50 ml M 4
- Feeding during test: no feeding during the test period

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

Not applicable.

Hardness:

200 mg/L CaCO3

Test temperature:

19-21 °C

pH:

7.89 (pH of test water)
For pH of test solutions at t= 0h and t= 48 h at different test concetrations see table 2 below.

Dissolved oxygen:

8.8 mg/L

Salinity:

not applicable

Nominal and measured concentrations:

Nominal: 0 (control), 0.99, 1.48, 2.22, 3.33, 5.00, 7.50, 11.25 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: glass beaker, volume 2000 ml
- Type (delete if not applicable): open
- Fill volume: 2000 ml
- Aeration: no
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4 (+1 for measuring)
- No. of vessels per control (replicates): 4 (+1 for measuring)

TEST MEDIUM / WATER PARAMETERS
Dilution water used in the test period was reconstituted water:
CaCl2: 2 mmol/l
MgSO4: 0.5 mmol/l
NaHCO3: 0.75 mmol/l
KCl: 0.075 mmol/l
The sum of calcium and magnesium ions in the dilution water was 2.5 mmol/l. The proportion of Ca : Mg ions was 4 : 1 and of Na : K ions 10 : 1. The acid capacity Ka 4.3 of the solution was 0.8 mmol/l.
Derived from the CaCl2 addition, the hardness of water was equal to 2 mmol/l CaCO3, which corresponds to a hardness of 200 mg/l CaCO3 (valid: 10 – 250 mg/l).

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: Light-dark-cycle: 16 h light : 8 h dark, daily
- Light intensity: no data

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): at 3, 24 and 48 hours after application: number of immobile Daphnia per vessel

Reference substance (positive control):

yes

Remarks:

potassium dichromate, batch No.: 060983, purity: 99.5 %, p.a.; dose rate in the test to control the sensitivity: 0.586...2.1 mg/l

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

4.62 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95 % C.I.: 4.10 -.5.21 mg/L

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

4.42 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95 % C.I.: 3..99 -.4.90 mg/L

Key result

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

2.22 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

2.18 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

3.33 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

3.4 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

Abnormal responses of the Daphnia:
At any time of inspection no visible signs of abnormalities of surviving Daphnia were observed.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- The reference substance potassium dichromate is tested periodically to control the sensitivity of the test system. The results of the last test (21.04.-23.04.98) are the following:
* EC50 (24 h) = 1.533 mg/L
* EC50 (48 h) = 1.273 mg/L

Reported statistics and error estimates:

The determination of the median lethal dose value (LD50) was carried out by Probit analysis (EASY ASSAY Critical Values Ver. 3.01, 1998 by RATTE) according to FINNEY (1971).
Regarding the NOEC and LOEC`s, the results were proved by means of multiple comparison procedure for comparing several treatments. This procedure was a variance test (ANOVA / Dunnett’s t-test), testing the significance of the differences between each of the treatments compared to the mean values of each group (5 treatments).

Table 1: Cumulative mortality at 3, 24 and 48 hours after application

		3 h					24 h					48 h
Calciumcarbide CaC2(nominal/measured)(technical product)	AcetyleneC2H2	after application
		Number of immobilised Daphnia/replicate
		1	2	3	4	Sum	1	2	3	4	Sum	1	2	3	4	Sum
Control	Control	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
0.99/1.04	0.40	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
1.48/1.50	0.60	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
2.22/2.18	0.90	0	0	0	0	0	0	0	0	0	0	0	0	1	0	1
3.33/3.40	1.35	0	0	0	0	0	0	0	0	0	0	1	0	1	1	3*
5.00/5.10	2.03	0	0	0	0	0	1	1	0	1	3*	3	2	2	2	9*
7.50/6.01	3.05	0	2	2	3	7*	3	4	3	4	14*	5	5	5	5	20*
11.25/7.74	4.56	5	5	5	5	20*	5	5	5	5	20*	5	5	5	5	20*
		Calciumcarbide mg/l (nominal/measured) / (Acetylene mg/l (nominal/measured))
EC50 (mg/l)							6.49/5.64 (2.64/2.29)					4.62/4.42 (1.88/1.79)
95 % confidence interval							5.78...7.18/5.35...5.96					4.10...5.21/3.99...4.90
slope							9.89/19.16					6.95/7.82
LOEC (mg/l)							5.0/5.1 (2.03/2.07)					3.33/3.4 (1.35/1.38)
NOEC (mg/l)							3.3/3.4 (1.35/1.38)					2.22/2.18 (0.90/0.88)

*:statistically significant, Dunnett-Test, p<0.05

The median effective concentration (EC50 after 48 hours) of Calciumcarbide, i.e. the concentration which proved immobilisation to 50 % of Daphnia was 4.62 mg/l Calciumcarbide (nominal conentration) and 4.42 mg/l Calciumcarbide (measured concentration).

The highest concentration tested at which the measured parameters did not show a significantly increased immobilisation of Daphnia relative to control values is considered the “No Observed Effect Concentration” (NOEC).

At the test concentration of 2.2 mg/l Calciumcarbide (nominal concentration) and 2.18 mg/l Calciumcarbide (measured concentration) no statistically significant increase in the number of immobilised Daphnia could be determined after 48 h, so this concentrations has to be set as NOEC for the nominal and measured concentrations.

The lowest concentration tested at which the measured parameters showed a significantly increased immobilisation of Daphnia relative to control values is considered the “Lowest Observed Effect Concentration” (LOEC). At the test concentration 3.33 mg/l Calciumcarbide (nominal concentration) and 3.4 mg/l Calciumcarbide (measured concentration) a statistically significant increase in the number of immobilised Daphnia could be determined after 48 h, so this concentration has to be set as LOEC.

Oxygen concentrations, pH values and temperatures

During the test period, the dissolved oxygen concentrations and the pH values of the control and of the test solutions were checked at the start of the test and after 48 hours.

1) Dissolved oxygen concentration:

- O2 concentration at the start of the test (t = 0), all test vesseld: 8.8 mg/L

- O2 concentration after 48 h, all test vessels: 8.6 mg/L

volumetric instrument: O2-meter OXI 96, WTW Wissenschaftlich-Technische Werkstätten GmbH, D-82362 Weilheim, Germany

2) pH value

Table 2:

Calciumcarbide (technical product)concentration (mg/l)	pH value
	0 h	48 h after application
Control	7.88	8.16
0.99	8.14	8.15
1.48	8.27	8.15
2.22	8.40	8.21
3.33	8.55	8.28
5.00	8.19	8.39
7.50	9.27	8.46
11.25	10.86	9.81

volumetric instrument: pH-meter pH 95, WTW Wissenschaftlich-Technische Werkstätten GmbH, D-82362 Weilheim, Germany.

The pH value of the test solutions was not adjusted.

Validity criteria fulfilled:

yes

Remarks:

- Number of immobile Daphnia which are immobilised or trapped at the surface of the water: /= 60 % of the air saturation value at the temperature used

Conclusions:

The 48-hr EC50 of calcium carbide to Daphnia magna is 4.62 mg/L (nominal concentration).

Executive summary:

The 48-hr-acute toxicity of calcium carbide to Daphnia magna was studied under static conditions. Daphnids were exposed to the test chemical at nominal concentrations of 0 (control), 0.99, 1.48, 2.22, 3.33, 5.00, 7.50, and 11.25 mg/L for 48 hr. Mortality/immobilization were observed at 3, 24 amd 48 h after start of the test. The 48- hour EC50 was 4.62 mg/L nominal. The 48 -hr NOEC based on mortality/immobilization was 2.22 mg/L nominal.

This study is classified as acceptable and satisfies the guideline requirements for an acute toxicity study with freshwater invertebrates.

Results Synopsis

Test Organism Age: 2 -24 h

Test Type: Static

EC50: 4.62 mg//L (95% C.I. : 4.10 to 5.21 mg./L)

NOEC: 3.33 mg/L

Endpoint(s) Effected: mobility

Description of key information

The 48-hr EC50 of calcium carbide to Daphnia magna is 4.62 mg/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

4.62 mg/L

Additional information

The 48-hr-acute toxicity of calcium carbide to Daphnia magna was studied under static conditions. Daphnids were exposed to the test chemical at nominal concentrations of 0 (control), 0.99, 1.48, 2.22, 3.33, 5.00, 7.50, and 11.25 mg/L for 48 hr. Mortality/immobilization were observed at 3, 24 and 48 h after start of the test. The 48- hour EC50 was 4.62 mg/L nominal. The 48 -hr NOEC based on mortality/immobilization was 2.22 mg/L nominal.

This study is classified as acceptable and satisfies the guideline requirements for an acute toxicity study with freshwater invertebrates.

Results Synopsis

Test Organism Age: 2 -24 h

Test Type: Static

EC50: 4.62 mg//L (95% C.I. : 4.10 to 5.21 mg./L)

NOEC: 3.33 mg/L

Endpoint(s) Effected: mobility