Reaction mass of calcium dihydroxide and calcium peroxide

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28/04/2018 - 25/05/2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum)

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):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata)
- Strain: NIVA CHL 1
- Source: In-house laboratory culture (Charles River Den Bosch Laboratories)
- Age of inoculum (at test initiation): 3 days.
- Method of cultivation: Cultured under standardised conditions (OECD 201)

ACCLIMATION
- Acclimation period: 3 days.
- Culturing media and conditions: same as test
- Any deformed or abnormal cells observed: no.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

24 mg/L (CaCO3)

Test temperature:

21 ± 1ºC

pH:

8.1 ± 0.2

Nominal and measured concentrations:

WAFs at loading rates of 1.0, 3.2, 10, 32 and 100 mg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL, all-glass, containing 50 mL of test solution
- Initial cells density: 1.0 E4 cells/ml
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Milli-RO water (tap-water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA)
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: continuously illuminated
- Light intensity and quality: TLD-lamps with a light intensity within the range of 76 to 79 µE.m-2.s-1.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: at the beginning of the teest, microscope and counting chamber; thereafter by spectrophotometer.
- Chlorophyll measurement: no.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study: the project started with a combined limit/range-finding test, but at the end of the test, algae were not observed to verify a normal and healthy appearance.
- Test concentrations: for the range finding study, WAFs at loading rates of 1.0, 10, and 100 mg/L; for the main study, WAFs at loading rates of 1.0, 3.2, 10, 32 and 100 mg/L were prepared.
- Results used to determine the conditions for the definitive study: A final test was performed based on the results of a preceding combined limit/range-finding test.

Reference substance (positive control):

yes

Remarks:

potassium dichromate (Merck, Art. 1.04864, Batch K44879664).

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

36 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: CL: 35 - 38 mg/L.

Key result

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

based on biological relevance.

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

3.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: CL: 3.4 - 3.7 mg/L.

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Details on results:

A dose-related increase of algal growth inhibition was observed with increasing loading rate, whereby the effects observed at loading rates of 10 and 32 mg/L were comparable. The inhibition of both growth rate and yield was statistically significant at all loading rates. The inhibition of growth rate observed at a loading rate of 1.0 mg/L was below 10% and thus considered to be biologically not relevant.

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells.
- Any stimulation of growth found in any treatment: no.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- EC50: 72h-EC50 = 1.6 mg/L (95% CL: 1.5 - 1.6 mg/L) based on growth rate.

Reported statistics and error estimates:

Probit analysis method for Percent Inhibition of Growth Rate. Calculation of ELx values was based on probit analysis using linear max. likelihood regression with the percentages of growth rate inhibition and the percentages of yield inhibition versus the logarithms of the corresponding loading rates of the test item. An effect was considered to be significant if statistical analysis of the data obtained for the test loading rates compared with those obtained in the negative control revealed significant inhibition of growth rate or inhibition of yield (Multiple Sequentially-rejective Welsh-t-test After Bonferroni-Holm, α=0.05, one-sided, smaller). Test for normality of distribution: Shapiro-Wilk's Test on Normal Distribution; test for homogeneity of variance: Levene's Test on Variance Homogeneity; determination of NOELR: Multiple Sequentially-rejective Welsh-t-test After Bonferroni-Holm.

Table 1. Mean Cell Densities (x10⁴ Cells/mL) During the Combined Limit/Range-Finding Test

Time (h)	Test item, WAF loading rate (mg/L)
	Control	1.0	10	100
0	1.0	1.0	1.0	1.0
24	11.6	n.d.	n.d.	1.7
48	65.1	n.d.	n.d.	1.0
72	276.9	271.1	75.3	1.2

* n.d.: not determined

 

Table 2. Percentage Inhibition of Growth Rate during the Combined Limit/Range-Finding Test

Test item WAF loading rate (mg/L)	Mean	Std. Dev.	n	%Inhibition
Control	1.874	0.0095	6
1.0	1.868	0.0014	3	0.37
10	1.440	0.0200	3	23
100	0.073	0.0302	6	96

 

Table 3. Percentage Inhibition of Yield during the Combined Limit/Range-Finding Test

Test item WAF loading rate (mg/L)	Mean	Std. Dev.	n	%Inhibition
Control	275.9	7.92	6
1.0	270.1	1.15	3	2.1
10	74.3	4.58	3	73
100	0.2	0.11	6	100

 

Table 4. Growth Rate and Percentage Inhibition for The Total Test Period

Test item WAF loading rate (mg/L)	Mean	Std. Dev.	n	%Inhibition
Control	1.778	0.0165	6
1.0	1.755	0.0009	3	1.3*#
3.2	1.440	0.0217	3	19*
10	1.313	0.0743	3	26*
32	1.321	0.0224	3	26*
100	0.000	0.0000	3	100*

* Effect was statistically significant. # Effect was biologically not relevant (< 10%).

Table 5. Growth Rate and Percentage Inhibition at Different Time Intervals

Test item WAF loading rate (mg/L)	n	0 – 24 h		24 – 48 h		48 – 72h
		Mean	%Inhibition	Mean	%Inhibition	Mean	%Inhibition
Control	6	1.891		1.824		1.619
1.0	3	1.885	0.31	1.764	3.3	1.617	0.16
3.2	3	1.746	7.7	1.568	14	1.005	38
10	3	1.899	-0.43	1.415	22	0.624	61
32	3	1.888	0.18	1.406	23	0.668	59
100	3	0.107	94	-0.09	105	-0.017	101

 

Table 6. Yield and Percentage Inhibition for the Total Test Period.

Test item WAF loading rate (mg/L)	Mean	Std. Dev.	n	%Inhibition
Control	206.4	10.44	6
1.0	192.6	0.54	3	6.7*#
3.2	74.3	4.98	3	64*
10	51.2	12.22	3	75*
32	51.6	3.61	3	75*
100	0.0	0.00	3	100*

* Effect was statistically significant. # Effect was biologically not relevant (<10%).

 

Table 7. Effect Parameters

	Parameter (mg/L)	NOEL*	NOEL**	EL10	EL20	EL50
Growth rate	Value	<1.0	1.0	9.4	15	36
	lower 95%-cl			8.6	14	35
	upper 95%-cl			10	16	38
Yield	Value	<1.0	1.0	0.44	0.90	3.5
	lower 95%-cl			0.39	0.82	3.4
	upper 95%-cl			0.50	0.98	3.7

cl – confidence limit; * based on statistical significance; ** based on biological relevance

Table 8. pH Levels Recorded During the Final Test.

Test item WAF loading rate (mg/L))	pH
	t=0h	t=72h
Control	8.5	8.4
1.0	8.5	8.3
3.2	8.5	8.2
10	8.5	8.0
32	8.5	8.0
100	8.4	8.0

 

Table 9. Overview of % inhibition of growth rate in the reference test:

Nominal conc. K2Cr2O7 (mg/L)	Mean	Std. Dev.	n	%Inhibition
Control	1.928	0.0338	3
0.18	1.930	0.0294	3	-0.064
0.32	1.837	0.0309	3	4.8
0.56	1.651	0.0908	3	14
1.0	1.225	0.0161	3	36
1.8	0.750	0.0030	3	61
3.2	0.607	0.0272	3	69

 

Table 10. Overview of % inhibition of yield in the reference test:

Nominal conc. K2Cr2O7 (mg/L)	Mean	Std. Dev.	n	%Inhibition
Control	325.6	32.36	3
0.18	326.6	28.33	3	-0.29
0.32	246.9	22.32	3	24
0.56	143.8	36.03	3	56
1.0	38.5	1.94	3	88
1.8	8.5	0.09	3	97
3.2	5.2	0.50	3	98

Potassium dichromate inhibited growth rate of this fresh water algae species at nominal concentrations of 0.32 mg/L and higher.

Validity criteria fulfilled:

yes

Remarks:

Cell concentration in the control cultures increased exponentially (factor of 207). The mean coefficient of variation in control cultures was 8.4%. Coefficient of variation of average specific growth rates in the replicate control cultures = 0.9%.

Conclusions:

The 72h-EL50 of the test item in green algae is 36 mg/L based on growth rate. The NOELR for growth rate inhibition was determined to be 1.0 mg/L based on biological relevance.

Executive summary:

A growth inbition test on Pseudokirchneriella subcapitata was performed with the test item according to the OECD Guideline 201 (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 the results of a preceding combined limit/range-finding test. Six exponentially growing algal cultures of P. subcapitata were exposed to an untreated control, whereas three replicates per group were exposed to WAFs prepared at loading rates of 1.0, 3.2, 10, 32 and 100 mg test item per litre. The initial algal cell density was 10^4 cells/mL.  The total exposure period was 72 hours. The algal cultures were assessed for the growth by visual cell count at 24, 48 and 72 h. No analysis of the test media was conducted because the development of an analytical method was not feasible. The study met all acceptability criteria and was considered valid. A concentration-effect relationship was observed and statistically analysed to obtain effect concentrations. The test item reduced growth rate and inhibited the yield of this fresh water algae species significantly at WAF prepared at loading rates of 1.0 mg/L and higher. The growth rate inhibition effect concentrations with their respective 95% lower and upper confidence limits for the inhibition of growth rate were ErL10 = 9.4 (8.6 - 10) mg/L, ErL20 = 15 (14 -16), ErL50 = 36 (35 - 38) mg/L. The yield inhibition effect concentrations with their respective 95% lower and upper confidence limits for the yield were EyL10 = 0.44 (0.39 - 0.50) mg/L, EyL20 = 0.90 (0.82 - 0.98) mg/L, EyL50 = 3.5 (3.4 - 3.7) mg/L. The 72h-NOELR for both growth rate and yield was 1.0 mg/L based on biological relevance.

Description of key information

Key study. Method according to OECD 201, GLP study. The test item has a 72h-ErL50 = 36 mg/L and 72h-NOEL = 1.0 mg/L in algae, based on mobility.

Key value for chemical safety assessment

EC50 for freshwater algae:

36 mg/L

EC10 or NOEC for freshwater algae:

1 mg/L

Additional information

Key study. Growth inbition test on Pseudokirchneriella subcapitata, performed according to the OECD Guideline 201 (GLP study). As the test item was not completely soluble in test medium at the loading rates initially prepared, WAFs 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 the results of a preceding combined limit/range-finding test. Six exponentially growing algal cultures (initial cell density 10^4 cells/mL) were exposed to an untreated control, whereas three replicates per group were exposed to WAFs prepared at loading rates of 1.0, 3.2, 10, 32 and 100 mg test item per litre. The algal cultures were assessed for the growth by visual cell count at 24, 48 and 72 h. No analysis of the test media was conducted. All acceptability criteria were met.

A concentration-effect relationship was observed and statistically analysed to obtain effect concentrations. Reaction mass of calcium dihydroxide and calcium peroxide reduced growth rate and inhibited the yield of this fresh water algae species significantly at WAF prepared at loading rates of 1.0 mg/L and higher. The growth rate inhibition effect concentrations with their respective 95% lower and upper confidence limits for the inhibition of growth rate were ErL10 = 9.4 (8.6 - 10) mg/L, ErL20 = 15 (14 -16), ErL50 = 36 (35 - 38) mg/L. The yield inhibition effect concentrations with their respective 95% lower and upper confidence limits for the yield were EyL10 = 0.44 (0.39 - 0.50) mg/L, EyL20 = 0.90 (0.82 - 0.98) mg/L, EyL50 = 3.5 (3.4 - 3.7) mg/L. The 72h-NOELR for both growth rate and yield was 1.0 mg/L based on biological relevance.