Calcium sulfide (Ca(Sx))

Toxicity to microorganisms

Administrative data

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))

Version / remarks:

22 July 2010

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Calcium sulfide (Ca(Sx))

EC Number:

215-709-2

EC Name:

Calcium sulfide (Ca(Sx))

Cas Number:

1344-81-6

Molecular formula:

CaSx (X=2,3,4,5,6,7)

IUPAC Name:

Calcium sulfide (Ca(Sx)

Test material form:

liquid

Specific details on test material used for the study:

Species:
Activated sludge, microorganisms from a domestic waste water treatment plant.
Origin:
The (controlled) activated sludge was supplied by the sewage plant for domestic sewage in Balatonfüred, Hungary, on 12 December 2017 (one day before the test).
Preparation of Activated Sludge Inoculum:
The coarse particles were removed by settling for 10 minutes, and the upper layer of finer solids was decanted. The activated sludge used for this study was washed by centrifugation and the supernatant liquid phase was decanted. The solid material was re-suspended in isotonic saline solution with shaking and again centrifuged. This procedure was repeated twice.
An aliquot of the final sludge suspension was weighed (5.807 g wet weight), dried and the ratio of wet sludge to dry weight (0.5368 g dry weight) determined. Based on this ratio, calculated amount of wet sludge (24 g dry weight that was equivalent to 259.63 g wet sludge) was suspended in isotonic saline solution (ad. 8 L) to yield a concentration equivalent to about 3 g per litre (on dry weight basis).
(In the test containers (300 mL final volume) the final concentration of suspended solids, containing 150 mL inoculum was 1.5 g per litre on dry weight basis.)
The above concentration calculation accounts for the dilution resulting from feeding with synthetic sewage. The activated sludge was not used on the day of the collection, but continuously aerated (2 L/minute) at the test temperature for about 24 hours (1 day) and fed daily with 50 mL synthetic sewage/L activated sludge.
The pH of the activated sludge inoculum was checked after preparation (pH: 7.46), additional pH adjustment of the inoculum was considered not necessary.

Foaming:
In this test the occurring foaming was not significant, controlling was not necessary during the incubation.

Test organisms

Test organisms (species):

activated sludge of a predominantly domestic sewage

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Conc. based on:

test mat.

Effect concentrations 2

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

> 1 000 mg/L

Effect concentrations 3

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

ca. 1 000 mg/L

Applicant's summary and conclusion

Validity criteria fulfilled:

no

Conclusions:

Validity of the Study

The specific respiration rate of the blank controls (without the test substance or reference substance) was 40.59 mg oxygen per one gram of activated sludge (dry weight of suspended solids) in an hour (higher than 20 mg/gh) with a coefficient of variation of 6.91 %.
The 3-hour EC50 of the reference item 3,5-Dichlorophenol (for the used activated sludge batch) was 16.63 mg/L within the range of 2 mg/L to 25 mg/L, that was required for total respiration (in this study the differentiation between heterotrophic respiration and nitrification was considered as not necessary).

Specific Respiration Rates in the Test Item Concentrations

Defined amounts of the test item (1 x 3, 1 x 30, 3 x 300 mg in the test item treatments and 3 x 300 mg in the abiotic controls) were directly added in each test container, and after a pH measurement inoculated. The test item was investigated up to and including the concentration of 1000 mg/L without any pH adjustment before the inoculum addition. Analytical verification of test concentrations was not carried out.

In the experiment abiotic oxygen consumption of the test substance was not observed (in average 0.22 mg/Lh, see Table 4); therefore the total oxygen consumption rates were not corrected with the abiotic control values in the subsequent calculations. The measured nitrification potential of the applied activated sludge (see nitrification control values) did not necessitate further testing. The influence of MÉSZKÉNLÉ on the oxygen consumption and specific respiration rate of activated sludge is presented in Tables 4-5.
The observed oxygen consumption rates and consequently the specific respiration rates were in the range of the blank controls. No inhibitory effect of the test item was observed. The observed -0.51 % at 10 mg/L, 3.83 % at 100 mg/L and in average 0.79 % at 1000 mg/L inhibitions were within the biological variability of the applied test system.
The specific respiration rates of the highest dose, 1000 mg/L were compared with the blank control values using 2 Sample t-Test (2-sided, α=0.05). No statistical significant differences were observed in the comparison with the blank control values. Based on the results of this study the NOEC was determined to be 1000 mg/L.
Based on measured oxygen consumption values and calculated specific respiration rates it is concluded that the 3-hour EC10 and EC50 values of the test item are higher than 1000 mg/L.
The EC50 value was determined as: EC50 > 1000 mg/L.

Toxicity of 3,5-Dichlorophenol

The following concentrations of the positive reference control 3,5-Dichlorophenol were tested on the same activated sludge and under identical conditions as the test item: 2, 7 and 24.5 mg/L. In comparison to the blank controls the oxygen consumption rate of the activated sludge was inhibited by 13.28 % at the lowest concentration of 2 mg/L and at the nominal concentrations of 7 and 24.5 mg/L, the oxygen consumption rate was inhibited by 26.38 % and 73.39 %, respectively. The results are summarized in Appendix I, Tables 4 and 5.
The 3-hour EC50 of 3,5-Dichlorophenol was calculated to be 16.63 mg/L.

Nitrification Controls

An additional nitrification control was examined in the test with three parallels to check the possible nitrification potential of the applied activated sludge batch. With the applying of the nitrification control the differentiation between the total, heterotrophic and nitrification respiration was possible. The total respiration (RT) was 60.89 mg/Lh, the heterotrophic respiration (RH) was 61.23 mg/Lh, the nitrification respiration (RN): 0.34 mg/Lh was calculated according to the equation: RN= RT-RH.
The obtained 0.34 mg/Lh was considered as not significantly different, being within a biological variability range of the applied test system, and lower than the 5 % of RT (3.04 mg/Lh) in blank controls, Appendix I, Tables 4 and 5.
According to the above calculations and based on the OECD 209 guideline it was assumed that the heterotrophic oxygen uptake equals the total uptake.

Environmental Conditions (pH, Temperature)

This test was performed without pH adjustment. The measured pH values in the prepared solutions: 3,5-Dichlorophenol reference control stock solution, synthetic sewage, activated sludge inoculum prepared test mixtures (controls, and in the case of test item at the concentrations of 10 and 100 mg/L) etc. were in the acceptable pH 7-8 range at the start of the test.
In the test item containing test mixtures higher pH values were measured at the start of the test, the test item had a significant effect on the pH at the highest concentration level examined (1000 mg/L). The pH in the test containers containing 3 and 30 mg test item – water - synthetic sewage (10 and 100 mg/L, see Table 2: T1 and T2) was pH ~ 7.7 and 7.9, both within the required range of pH 7-8.
The pH in the test containers containing 300 mg test item - water – synthetic sewage (1000 mg/L, see Table 2: T3A-T3C, CA1-CA3, test item treatments and abiotic controls) was ~9 and ~8.6 after inoculation (test item treatments); both out of the required 7—8 pH range.
The test item may adversely affect pH within the test system; however the results of the present preliminary test show, that the higher pH caused by the test item has no effect on the applied inoculum culture and further testing (a definite test) with additional neutralization step of test item containing mixtures before inoculum addition is not necessary.
The measured pH values, the pH changes are summarized in Appendix I, Table: 3.

The mixtures were aerated at 0.5 L/min in the temperature range of 20±2oC. The temperature in the test mixtures during the measurements in average: 21.45oC, the measured minimum: 20.0 oC, maximum: 22.0 oC.

Executive summary:

Under the conditions of the performed Activated Sludge Respiration Inhibition Test, the EC₁₀and EC₅₀values of test item were determined as higher than 1000 mg/L. Based on the statistical evaluation in this test the NOEC was 1000 mg/L. In conclusion this preliminary test demonstrated the absence of inhibition of oxygen consumption by the test substance up to and including the limit concentration of 1000 mg/L. Therefore, in line with OECD 209, a definite test is not required.