Calcium hydrosilicate, reaction product of natural quartz sand and technical lime by a hydrothermal and tribochemical process

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:

2014-04-01 to 2014-03-31

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: The water accommodated fraction (WAF) was prepared with a nominal loading of the test item of 100 mg/L.
- Sampling method: The concentrations of the test item were analytically determined from the limit loading, the pH-control and the control after 0 hours (test start) and 72 hours (test end) via photometric analysis. Samples at test start were taken from an additional replicate without algae. At the end of the test samples were taken from pooled replicate with algae.
- Sample storage: All original samples were stored in a refrigerator until sample preparation. Prepared samples were stored at room temperature until analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The water accommodated fraction (WAF) was prepared with a nominal loading of the test item of 100 mg/L.
100 mg/L of the test item were weighed out and transferred with an appropriate amount of dilution water into a brown glass flask. This dispersion was shaken for 24 hours with 20 rpm at room temperature. After a separation phase of 1 hour, the aqueous phase or WAF was removed by siphoning (from the approximate centre of the glass flask) to give the 100 mg/L loading rate WAF.
- Eluate: Dilution water
- Differential loading: None, limit test
- Controls: Six replicates (without test item) were tested under the same test conditions as the test vessels.
- pH-Controls: Six replicates, neutralized to pH 8.1 ± 0.2 with NaOH/HCl

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Desmodesmus subspicatus Chodat
- Strain: SAG 86.81
- Source (laboratory, culture collection): SAG Pflanzenphysiologisches Institut der Universitaet Goettingen, Nikolausberger Weg 18, D-37073 Goettingen, Germany
- Age of inoculum (at test initiation): 4 days
- Method of cultivation: Fresh stocks were prepared every month on Z-Agar. Light intensity amounted to 35 - 70 µE x m-2 x s-1 for 24 h per day.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

No

Hardness:

not specified

Test temperature:

min.: 22.0 max.: 23.0, mean value: 22.5 °C

pH:

Nominal test item loading pH-value
[mg/L] Start; 0 hours End; 72 hours
100 10.58 8.38
100, pH-control 8.13 8.48
Control 8.18 8.52

Dissolved oxygen:

Not measured

Salinity:

Not measured, freshwater conditions

Nominal and measured concentrations:

See window "Any other information on materials and methods incl. tables"

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks
- Type (delete if not applicable): open, cotton wool plugs
- Material, size, headspace, fill volume: sterile 250 mL Erlenmeyer flasks, test volume 100 mL
- Aeration: Test containers were placed on a rotary shaker and oscillated at appr. 70 rpm
- Type of flow-through (e.g. peristaltic or proportional diluter): None
- Renewal rate of test solution (frequency/flow rate): One application at test start
- Initial cells density: Nominal: approximately 2 - 5 x 10^3 cells/mL, Actual: mean 4998 cells/mL
- Control end cells density: Mean 920143 cells/mL
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6
- No. of vessels per pH-control (replicates): 6



GROWTH MEDIUM
- Standard medium used: yes



TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
Composition of Dilution Water
according to the guideline
Component Concentration [mg/L]
NH4Cl 15
MgCl2 x 6 H2O 12
CaCl2 x 2 H2O 18
MgSO4 x 7 H2O 15
KH2PO4 1.6
FeCl3 x 6 H2O 0.064
Na2EDTA x 2 H2O 0.1
H3BO3 0.185
MnCl2 x 4 H2O 0.415
ZnCl2 3 x 10^-3
Na2MoO4 x 2 H2O 7 x 10^-3
CoCl2 x 6 H2O 1.5 x 10^-3
CuCl2 x 2 H2O 1 x 10^-5
NaHCO3 50
pH 8.1 ± 0.2



OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: yes, 8.1 ± 0.2 for pH-control samples
- Photoperiod: 24 h
- Light intensity and quality: 4440 - 8880 lux, corresponding to 60 - 120 µE x m^-2 x s^-1, mean value: 5225 lux



EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter] fluorimeter, Cell density and self fluorescence was measured daily via Chlorophyll-a- fluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as background signal.



TEST CONCENTRATIONS

Results of the Range Finding Test (0 - 72 hours)
Nominal Test Item Loading
[mg/L] Growth Rate Inhibition [%] Yield Inhibition [%]
100 (pH adjusted) 0 1
100 2 12
10 4 20
1 6 27

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

< 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test):
- Unusual cell shape: No
- Colour differences: No
- Flocculation: No
- Adherence to test vessels: No
- Aggregation of algal cells: No
- Other: No
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No
- Effect concentrations exceeding solubility of substance in test medium: No

Results with reference substance (positive control):

- Results with reference substance valid? Yes

Current Study Valid Range (average ± 3 x SD)
Growth Rate Inhibition
ErC50 0.684 0.684 ± 0.281
95 % confidence interval 0.657 - 0.714 0.684 ± 0.281
Yield Inhibition
EyC50 0.315 0.314 ± 0.087
95 % confidence interval 0.297 - 0.3355 0.314 ± 0.087

SD = Standard deviation

Reported statistics and error estimates:

NOEL/LOEL were determined by calculation of statistical significance of growth rate and yield. As standard One Way Analysis of Variance (ANOVA) and DUNNETT’s test were used for NOEL/LOEl calculations. When running a One Way Analysis of Variance a Normality test and an Equal Variance test were done first. The SHAPIRO-WILK-Test was used to test for normally distributed populations. The Levene median test was used for equal variance. P-values for both Normality and Equal Variance tests are 0.05. The alpha-value (acceptable probability of incorrectly concluding that there is a difference) is alpha=0.05.

Cell Densities

Nominal Test Item Loading	Replicate	Cell Density [cells/mL]
[mg/L]	No.	0 hours	24 hours	48 hours	72 hours
100	1	4998	26587	164666	719393
	2	4998	21181	150745	639043
	3	4998	22970	139301	641815
	4	4998	22216	148224	637571
	5	4998	20730	151057	664642
	6	4998	22155	167170	665162
	Mean	4998	22640	153527	661271
100 neutralized (pH-control)	1	4998	37017	193159	884813
	2	4998	30164	192154	774316
	3	4998	35964	194865	727233
	4	4998	31741	166321	756947
	5	4998	34704	200713	729009
	6	4998	30017	183075	681839
	Mean	4998	33268	188381	759026
Control	1	4998	37229	182304	908159
	2	4998	30654	199552	835217
	3	4998	27877	221097	976857
	4	4998	29211	200427	931636
	5	4998	27812	191530	883470
	6	4998	32638	205495	985520
	Mean	4998	30904	200068	920143

Evaluation after 72 hours

                   Statistically significant differences of growth rates and yield compared to

                   control values are marked (+), not significant differences are marked (-).

Nominal Test Item Loading	Replicate	Growth Rate		Growth Rate Inhibition	Yield		Yield Inhibition
[mg/L]	No.	[d^-1]		[%]	[cells/mL]		[%]
100	1		1.66	5		714395	22
	2		1.62	7		634045	31
	3		1.62	7		636817	30
	4		1.62	7		632573	31
	5		1.63	6		659644	28
	6		1.63	6		660164	28
	Mean	(+)	1.63	6	(+)	656273	28
100 neutralized (pH-control)	1		1.73	1		879815	4
	2		1.68	3		769318	16
	3		1.66	4		722235	21
	4		1.67	4		751949	18
	5		1.66	4		724011	21
	6		1.64	6		676841	26
	Mean	(+)	1.67	4	(+)	754028	18
Control	1		1.73			903161
	2		1.71			830219
	3		1.76			971859
	4		1.74			926638
	5		1.73			878472
	6		1.76			980522
	Mean		1.74			915145

Section-by-Section and Average Specific Growth Rates of the Control Group
                   (0 – 72 hours)

	Replicate No.	Specific Growth Rate [d^-1]			Mean (0 - 72 h)	SD ±	CV [%]	Mean CV [%]
		Section-by-Section
		0 - 24 hours	24 - 48 hours	48 - 72 hours
Control	1	2.01	1.59	1.61	1.73	0.237	13.7	12.8
	2	1.81	1.87	1.43	1.71	0.240	14.0
	3	1.72	2.07	1.49	1.76	0.295	16.8
	4	1.77	1.93	1.54	1.74	0.196	11.2
	5	1.72	1.93	1.53	1.73	0.201	11.6
	6	1.88	1.84	1.57	1.76	0.169	9.58
				Mean	1.74
				SD ±	0.02
				CV [%]	1.20

Validity criteria fulfilled:

yes

Conclusions:

In this study celitement was found slightly to inhibit the growth rate of the freshwater green alga Desmodesmus subspicatus after 72 hours. The NOEC-value of the test item for both, inhibition of growth rate and inhibition of yield, after 72 hours was < 100 mg/L. The EC50-values of the test item for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were both > 100 mg/L, respectively.
All effect levels are given based on the nominal concentration of celitement.

Executive summary:

The toxicity of celitement to the unicellular freshwater green alga Desmodesmus subspicatuswas determined according to the principles of OECD 201 (2011) at Dr.U.Noack-Laboratorien in 31157 Sarstedt, Germany from2014-03-31 to 2014-04-04 with the definitive exposure phase from 2014-04-01 to 2014-04-04. The aim of the study was to assess the effects on growth rate and yield over a period of 72 hours.

 

The test item is a mixture, solid and insoluble in water. In view of the difficulties associated with the evaluation of aquatic toxicity of complex mixture test items, a modification of the standard method for the preparation of aqueous media was performed. An approach endorsed by several important regulatory authorities in the EU and elsewhere (ECETOC 1996 and OECD 2000), is to expose the organisms to a Water Accommodated Fraction (WAF*) of the test item in cases where the test item is a complex mixture. Exposures are expressed in terms of the original concentration of the test item in water at the start of the mixing period (loading rate) irrespective of the actual concentration of the test item in the WAF.

 

The study was conducted under static conditions with an initial cell density of 4998 cells/mL. One limit loading of 100 mg/L of the test item was prepared. The test item was applied onto a glass slide. The glass slide with the test item was inserted in a brown glass flask with an appropriate amount of dilution water. This dispersion was shaken for 24 hours with 20 rpm at room temperature.After a separation phase of 1 hour,the aqueous phase or WAF was removed by siphoning (from the approximate centre of the glass flask) to give the 100 mg/L loading rate WAF. The water soluble fraction was clear. Six replicates were tested for the limit loading, the pH - neutralized limit loading (pH – control) and for the control. Environmental conditions were within the acceptable limits.

 

The concentrations of the test item celitement were analytically evaluated via photometric analysis of silicon at the start of the exposure (0 h) and at the end of the exposure (72 h) of the limit loading level, the pH-control and the control.

The measured silicon concentration at the start of the exposure (0 h) was 7.45 mg/L in the limit loading level and 7.65 mg/L in the pH-control. At the end of the exposure the measured silicon concentrations gave recoveries of 98 and 102 % of the initially measured values of the limit loading and the pH-control indicating the stability of the test item in the WAF during the exposure phase.All effect values given are based on the nominal test item loading of celitement.

* Water accommodated fractions (WAF): The aqueous medium containing only the fraction of a multi-component test item that is dissolved and / or present as a stable dispersion/emulsion under test conditions in the test medium, acc. to OECD Guidance Document, No. 23.

Description of key information

Short-term toxicity testing with the freshwater green alga Desmodesmus subspicatus was carried out according to the OECD 201 guidance taking into account GLP and thus resulting in a Klimish 1 score. No acute toxicity was observed.

Additional information

The NOEC-value of the test item for both, inhibition of growth rate and inhibition of yield, after 72 hours was < 100 mg/L. The EC50-values of the test item for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were both > 100 mg/L, respectively.