Reaction mass of calcium dihydroxide and calcium peroxide

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28/04/2018 - 24/05/2018

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

ISO 6341 (Water quality - Determination of the Inhibition of the Mobility of Daphnia magna Straus (Cladocera, Crustacea))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Remarks:

not feasible.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Preparation of test solutions started with loading rates individually prepared in the range of 1.0 and 100 mg/L. A two-day period of magnetic stirring was applied to ensure maximum dissolution of the test item in test medium. Thereafter, the aqueous Water Accommodated Fractions (WAF’s) were collected by filtration through a 0.45 µm membrane filter (RC55, Whatman) and used as test concentrations. In the final test, the pH of the WAF prepared at a loading rate of 1.0 mg was adjusted from 8.0 to 8.5 with 1N NaOH (Merck, Darmstadt, Germany). The pH of the WAFs prepared at loading rates in the range of 3.2 to 100 mg/L were adjusted from 8.8-11.4 to 8.2-8.5 with 0.1 and 1.0 N HCL (Merck, Darmstadt, Germany).
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): no. All test solutions were clear and colorless at the end of the preparation procedure.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Strain/clone: Daphnia magna (Crustacea, Cladocera) (Straus, 1820)
- Source: In-house laboratory culture with a known history. Daphnids were at least third generation, obtained by a cyclical parthenogenesis under specified breeding conditions.
- Age of parental stock (mean and range, SD): newborn daphnids, i.e. less than 3 days old.
- Feeding during test: no.

ACCLIMATION
- Acclimation period: No, culture conditions same as test.
- Health during acclimation (any mortality observed): Daphnids originated from a healthy stock, 2nd to 5th brood, showing no signs of stress such as mortality >20%, presence of males, ephippia or discoloured animals were used for the study and there was no delay in the production of the first brood.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

180 mg/L as CaCO3

Test temperature:

18 - 22ºC

pH:

7.7 ± 0.3

Dissolved oxygen:

8.5 - 9.1 mg/L O2

Nominal and measured concentrations:

WAFs prepared at loading rates of 1.0, 1.8, 3.2, 5.6 and 10 mg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: 60 mL, all-glass, containing 50 mL test solution.
- Aeration: no.
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: tap water purified by Reverse Osmosis (RO-water, GEON Waterbehandeling, Berkel-Enschot, The Netherlands)
- Culture medium different from test medium: no.

OTHER TEST CONDITIONS
- Adjustment of pH: no.
- Photoperiod: 16 hours photoperiod daily

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Immobility (including mortality), at 24 hours and at 48 hours for all concentrations; pH an dissolved oxygen, at the beginning and at the end of the test, for all loading rates and the control; and temperature of medium, at the beginning at the start of the test, plus continuously in a temperature control vessel.

RANGE-FINDING STUDY
- Test concentrations: for the range finding study, WAFs at loading rates of 1.0, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: At the end of the preliminary test, complete immobility was observed at the WAFs prepared at loading rates of 10 and 100 mg/L. Based on these results, WAF’s individually prepared at loading rates of 1.0, 1.8, 3.2, 5.6 and 10 mg/L were used in the main study.

Reference substance (positive control):

yes

Remarks:

potassium dichromate (Merck, Darmstadt, Germany, Art. 1.04864, Batch no. K44879664, ).

Key result

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

8.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CL: 7.5 - 10.3 mg/L

Duration:

24 h

Dose descriptor:

EL50

Effect conc.:

> 10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

- No immobility was observed in the control or the three lowest test loading rates throughout the test. At the end of the test, 10% immobility was observed at a loading rate of 5.6 mg/L and 65% immobility was observed at the highest loading rate.
- Behavioural abnormalities: no.
- Mortality of control: no.

Results with reference substance (positive control):

- Results with reference substance valid? yes.
- Limit test: no.
- ECx: 24h-EC50 = 0.80 mg/L (95% CL: 0.71 - 0.90 mg/L); 48h-EC50 = 0.33 mg/L (95% CL: 0.29 - 0.38 mg/L).
- Relevant effect levels: the actual responses in this reference test were within the ranges of the expected responses at the different concentrations. Hence, the sensitivity was in agreement with the historical data collected.

Reported statistics and error estimates:

The 48h-EL50-value was calculated from the Probits of the percentages of affected daphnids and the logarithms of the corresponding test item loading rates using the maximum likelihood estimation method. The 24h-EL50 could not be determined because the observed effects were below 50%.

Table 1. Number of Introduced Daphnids and Incidence of Immobility in the Combined Limit/Range-Finding Test. 

Time (h)	Replicate	Test item, WAF loading rate (mg/L)
		Control	1.0	10	100
0	A	5	5	5	5
	B	5	5	5	5
	C	5			5
	D	5			5
	Total introduced	20	10	10	20
24	A	0	0	3	5
	B	0	0	5#	5
	C	0			5
	D	0			5
	Total immobilised	0	0	8	20
	Effect %	0	0	80	100
48	A	0	0	5	5
	B	0	0	5	5
	C	0			5
	D	0			5
	Total immobilised	0	0	10	20
	Effect %	0	0	100	100

# Microscopic observation revealed no test item attached to the daphnids.

 

Table 2. Number of Introduced Daphnids and Incidence of Immobility in the Final Test.

Time (h)	Replicate	Test item, WAF loading rate (mg/L)
		Control	1.0	1.8	3.2	5.6	10
0	A	5	5	5	5	5	5
	B	5	5	5	5	5	5
	C	5	5	5	5	5	5
	D	5	5	5	5	5	5
	Total introduced	20	20	20	20	20	20
24	A	0	0 (1)	0 (1)	0 (4)	0 (2)	0 (5)
	B	0	0 (1)	0 (2)	0 (2)	0 (2)	0 (5)
	C	0	0 (3)	0 (2)	0 (4)	0 (3)	0
	D	0	0 (1)	0 (3)	0 (2)	0 (1)	0 (2)
	Total immobilised	0	0	0	0	0	0
	Effect %	0	0	0	0	0	0
48	A	0	0	0 (1)	0 (1)	0 (2)	5#
	B	0	0	0	0	1 (2)	3 (1)
	C	0	0	0	0 (2)	1 (1)	1
	D	0	0	0	0	0	4
	Total immobilised	0	0	0	0	2	13
	Effect %	0	0	0	0	10	65

( ) between brackets: number of daphnia observed trapped at the surface of the test solutions. These organisms were reimmersed into the respective solutions before recording of mobility. # Microscopic observation revealed no test item attached to the daphnids. 

 

Table 3. Effect Parameters.

Parameter	Test item WAF loading rate (mg/L)	95%-confidence interval (mg/L)
24h-EC50	>10
48h-EC50	8.7	7.5 – 10

  

Table 4. pH and Oxygen Concentrations (mg/L) During the Final Test.

Test item WAF loading rate (mg/L)	Start (t=0 h)		End (t=48 h)
	pH	O2	pH	O2
Control	8.0	8.7	8.0	9.0
1.0	8.1	8.6	8.0	9.0
1.8	8.2	8.6	8.0	8.9
3.2	8.4	8.6	8.0	9.0
5.6	8.3	8.5	8.0	9.0
10	8.3	8.5	8.0	9.1

  

Table 5. Acute immobilization of daphnia after 24 and 48 hours in the reference test with potassium dichromate. 

Concentration K2Cr2O7	Number Exposed	% immobile 24h                     48h		Expected response (%) After 48 hours^1
(mg/L)				Minimal            Maximal
Control	20	0	0	0	10*
0.10	20	0	0	0	10
0.18	20	0	5	0	10
0.32	20	0	35	0	30
0.56	20	5	100	0	100
1.0	20	85	100	40	100
1.8	20	100	100	100	100

1: Based on historical data of the previous years (n>60). *A maximum response of 10% does not invalidate the results of the test.

Validity criteria fulfilled:

yes

Remarks:

In the control, no daphnids became immobilised or showed other signs of disease or stress; the oxygen concentration at the end of the test was ≥ 3 mg/L in control and test vessels.

Conclusions:

The 48h-EL50 of the test item in daphnia magna is 8.7 mg/L, based on mobility.

Executive summary:

An Acute Daphnia magna Immobilisation Test was performed with the test item, according to OECD Guideline 202 (GLP study). As the test item was not completely soluble in test medium at the loading rates initially prepared, Water Accomodated Fractions were individually prepared at loading rates ranging between 1.0 and 100 mg/L and used as test concentrations. A final test was performed based on a preceding combined limit/range-finding test. Twenty daphnids per group (5 per replicate, quadruplicate) were exposed to an untreated control and to WAFs prepared at loading rates of 1.0, 1.8, 3.2, 5.6 and 10 mg/L. The total exposure period was 48 hours. The study was performed without analytical confirmation of actual exposure concentrations because developing an analytical method was not feasible. The study met all acceptability criteria and was considered valid. No immobility was observed in the control or the three lowest test loading rates throughout the test. At the end of the test, 10% immobility was observed at a loading rate of 5.6 mg/L and 65% immobility was observed at the highest loading rate. In conclusion, the 48h-EC50 for Daphnia magna exposed to the test item was 8.7 mg/L based on loading rates (95% confidence interval between 7.5 and 10 mg/L).

Reference 2

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The study was carried out in accordance with US EPA testing guidelines. The determination of hydrogen peroxide concentrations in the test solutions was done using a semi-quantitative method.

Qualifier:

according to guideline

Guideline:

other:

Principles of method if other than guideline:

Other method: USEPA Toxic Substances Control Act Test Guidelines (1985), Revision of TSCA Guidelines (1987) and USEPA Methods of Measuring the Acute Toxicity of Effluents to Freshwater and Marine Organisms (1984).

GLP compliance:

no

Analytical monitoring:

yes

Details on sampling:

Test medium samples of all test substance groups were analysed at the beginning and at the end of each 24-hour test medium renewal interval.

Vehicle:

no

Details on test solutions:

Primary stock solutions (either 1 or 2 % w/v) were prepared immediately prior to bioassay initiation by volumetric dilution of weighed samples of a 50 % hydrogen peroxide solution. The reconstituted dilution water was prepared with distilled water/lake water 1:1 mixture (Lake Cammack, North Carolina). Stock solutions were stirred in their loosely stoppered volumetric flasks until well mixed. Then aliquot were weithdrawn for immediate concentration confirmation. Test solutions were prepared by dilution of the stocks.

Test organisms (species):

Daphnia pulex

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia pulex
- Strain: Freshwater waterflea (Daphnia pulex Leydig emend. Richard)
- Source: In-house breeding from a starting population obtained from the Aquatic Toxicology Group, Water Quality Section, North Carolina Quality Section, North Carolina Division of Enviromental Management, Raleigh
- Age at study initiation (mean and range, SD): 12 - 24 hours
- Food type: Greenalgae Selenastrum capricornutum, suspension of digested salmon chow, yeast and CEROPHYLL
- Photoperiod: Cultures are illuminated by ambient fluorescent laboratory light (500 lux) supplemented by a single fluroescent tube placed 30 cm above the cultures. A 16 hours light: 8 hours darkness photoperiod is maintained.
- No. of test animals: 10 per vessel

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

Not applicable

Hardness:

50 - 250 mg/L as CaCO3

Test temperature:

20 ± 1°C

pH:

Approximately neutral (not specified in more detail in the report)

Dissolved oxygen:

≥ 91.3 % of saturation

Salinity:

Not applicable

Nominal and measured concentrations:

Nominal concentration series: 1, 10, 50, 100, 500 and 1000 ppm
Measured concentration series (average): 1, 5.5, 23.6, 43.5, 246.9 and 489.5 ppm

Details on test conditions:

No. of vessels per concentration: 1
Controls: 2 controls

Reference substance (positive control):

no

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

1 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

dissolved

Basis for effect:

mortality

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

2.4 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

dissolved

Basis for effect:

mortality

Details on results:

Death was defined as the cessation of motion of the body or appendages.

Results with reference substance (positive control):

Data from sensitivity checks with reference toxicants are not shown in the report.

Reported statistics and error estimates:

LC50 values and the 95 % confidence limits were calculated using the Trimmed Spearman-Karber method.

Table 1: Experimental data from the daphnid hydrogen peroxide bioassay

Concentration of hydrogen peroxide (mg/L) determined by titration				Mortality: no. dead/no. tested		Spearman-Karber estimates
Nominal (planned)	Initial	Final	Mean	24 hours	48 hours	Data/LC50 (mg/L)
0.5 (1.0)	1.5	0.5	1.0	0/20	0/20	Nominal 3.2
5.0 (10.0)	6.5	4.6	5.5	20/20	20/20	--
25.0 (50.0)	24.8	22.4	23.6	20/20	20/20	Initial 3.1
50.0 (100.0)	48.9	38.2	43.5	20/20	20/20	--
250.0 (500.0)	246.9	246.9	246.9	20/20	20/20	Final 1.5
500.0 (1000.0)	489.9	489.1	489.5	20/20	20/20	--
--						Mean 24h: 2.4
						Mean 48h: 2.4

Validity criteria fulfilled:

no

Conclusions:

The acute toxicity of hydrogen peroxide to Daphnia pulex was tested and the LC50 value was 2.4 mg/L.

Executive summary:

The acute toxicity of hydrogen peroxide to invertebrates was tested in a semi-static, 48 - hours assay with Daphnia pulex according to USEPA Toxic Substances Control Act Test Guidelines (1985, 1987). Tested hydrogen peroxide concentrations ranged from 0.5 to 500 mg/L. The validity criteria of the test were fulfilled in the sense that no mortality was observed in the controls. Significant dissipation of the test material ranging from 10 to 60 % occurred within 24 hours with the four lowest test concentrations (0.5 to 50 mg/L, nominal). The LC50 value was determined to be 2.4 mg/L in the test. No reliable confidence intervals could be calculated. The NOEC value was 1 mg/L.

Description of key information

Key study. Method according to OECD 202, GLP study. The test item has a 48h-EC50 = 8.7 mg/L in daphnid, based on mobility.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

8.7 mg/L

Additional information

An Acute Daphnia magna Immobilisation Test was performed with the test item "reaction mass of calcium deihydroxide and calcium peroxide" according to OECD Guideline 202 (GLP study). As the test item was not completely soluble in test medium at the loading rates initially prepared, Water Accomodated Fractions were individually prepared at loading rates ranging between 1.0 and 100 mg/L and used as test concentrations. A final test was performed based on a preceding combined limit/range-finding test. Twenty daphnids per group (5 per replicate, quadruplicate) were exposed to an untreated control and to WAFs prepared at loading rates of 1.0, 1.8, 3.2, 5.6 and 10 mg/L. The total exposure period was 48 hours. The study was performed without analytical confirmation of actual exposure concentrations. The study met all acceptability criteria and was considered valid. No immobility was observed in the control or the three lowest test loading rates throughout the test. At the end of the test, 10% immobility was observed at a loading rate of 5.6 mg/L and 65% immobility was observed at the highest loading rate. In conclusion, the 48h-EC50 for Daphnia magna exposed to the test item was 8.7 mg/L based on loading rates (95% confidence interval between 7.5 and 10 mg/L).

Furthermore, supporting information is available on hydrogen peroxide (H2O2), the primary hydrolytic degradation products of “reaction mass of calcium dihydroxide and calcium peroxide”.

As evidenced in a water solubility study, the reaction mass goes into the chemical reactions below in water rapidly (i.e. within 24 hours) and hydrogen peroxide subsequently occurred.

 - CaO2 + 2 H2O -> Ca2 + + 2 (OH)- + H2O2

- H2O2 + H2O -> 2 H2O + O2

- Ca(OH)2 -> Ca2 + + 2 OH

The acute toxicity of hydrogen peroxide to Daphnia pulex was tested in a 48h experiment according to US EPA TSCA guidelines. The 48h-LC50value determined from this study was 2.4 mg/L.

From this value, a corresponding  48h-LC50 value can be calculated for the reaction mass by considering the hydrolytic reaction involved in the formation of hydrogen peroxide from calcium peroxide and the composition of the reaction mass (i.e. the reaction mass contains ca. 80% calcium peroxide). The amount of hydrogen peroxide generated binds in equimolar ratio to the amount of calcium peroxide.

Accordingly, 100 mg of the reaction mass contains 80 mg of calcium peroxide, which corresponds to 1.11 mmol of calcium peroxide available for the hydrolytic reaction. Thus, 1.11 mmol (= 37.74 mg) of hydrogen peroxide is formed upon hydrolysis of 100 mg of calcium peroxide.

Therefore, the 48h-LC50 value for the reaction mass, when calculated from the 48h-LC50 value for hydrogen peroxide (2.4 mg/L) is as follows:

- 100 mg of the reaction mass yields 37.74 mg hydrogen peroxide.

- 6.36 mg of the reaction mass yields 2.4 mg hydrogen peroxide.

The calculated 48h-LC50 value was comparable with the value experimentally determined in the study conducted on the reaction mass. This result indicates that the aquatic toxicity observed for the reaction mass can be attributed to the adverse effects exerted by the hydrogen peroxide formed upon hydrolysis of calcium peroxide and such effects can be predicted based on test data available for hydrogen peroxide.