Barium carbonate

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Samples from stock solution, and from test solutions of selected concentration levels were taken to determine the actual test item concentrations in comparison to the nominally applied concentrations. Samples of all concentrations were taken at the start of the test. Samples of the control, the lowest and the highest test concentration were taken at the end of the exposure period. Test solutions taken at the end of the exposure period were filtered using folded paper filters (pore size: 4 to 7 µm) to remove the microbial inoculum. The filtrates were sent to the Analytical Test Site for determination of the test concentrations. The sample volume required for analysis was approximately 100 mL per sample.

Vehicle:

no

Details on test solutions:

Reconstituted water according to OECD Guideline No. 203 (Fish, Acute Toxicity Test) was used to wash and to maintain the activated sludge. The reconstituted water contained salts at the following final concentrations:
- CaCl2 * 2 H2O: 294.0 mg/L
- MgSO4 * 7 H2O: 123.25 mg/L
- NaHCO3: 64.75 mg/L
- KCl: 5.75 mg/L

Preparation of stock solution and test solutions:
- A stock solution was prepared by dissolving 4.1298 g of the test item in 2000 mL of deionised water (nominal concentration 2.065 g/L). The stock solution was stirred on a magnetic stirring device until each single test vessel was prepared.
- The test item concentration levels were prepared by combining 16 ml of synthetic sewage feed with corresponding amounts of temperature adapted deionised water and/or stock solution in a way that a volume of 300 ml was obtained. Afterwards 200 ml of the microbial inoculum were added.
- The controls were prepared by combining 16 ml of the synthetic sewage feed with 284 ml of temperature adapted deionised water and adding 200 ml of the microbial inoculum.
- The total mixture volume for each of the test item concentrations and the controls was 500 mL per vessel. Immediately after preparation the test solutions were aerated.
- The test vessels were prepared at intervals of 15 minutes, starting with the first control, followed by the test item concentration levels. Replicates C6B (highest concentration level) and C0B (second control replicate exposed at the end of the testing series) were prepared 17 minutes after the preceding vessel to ensure equal exposure periods (3 hours) and maximum measurement periods (up to 10 minutes) for all vessels when testing the increased number of 14 test vessels.
- At the end of the contact time (test period), the content of the first test vessel (control 1) was poured into the measuring apparatus (consisting of oxygen-meter, flat bottom flask and stirring device) and the respiration rate was determined at two-second intervals over a period of up to 10 minutes. All other test vessels were measured according to this procedure in the same order which was established for the preparation of the test vessels.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

Activated sludge from a sewage treatment plant was used as the microbial inoculum for the test. The activated sludge was obtained from the sewage treatment works in Frankfurt/Main Niederrad treating predominantly domestic sewage.
- Date of sampling/ date of arrival at ECT: March 08, 2010
- Washing medium for activated sludge:Reconstituted water (OECD 1992)
- Frequency of washing: three times; between washings the activated sludge was centrifuged for 2 minutes at 1000 rpm
- Storage time before use: 4 days
- Storage temperature: 20 ± 2 °C

During storage 50 mL synthetic sewage feed were added per litre activated sludge and per day. The activated sludge was stirred and aerated during storage. The synthetic sewage feed consisted of the following amounts of substances in 1 litre of deionised water (in g/L):
- Peptone: 16.0
- Meat extract: 11.0
- Urea: 3.0
- NaCl: 0.7
- CaCl2 * 2 H2O: 0.4
- MgSO4 * 7 H2O: 0.2
- K2HPO4: 2.8
The required amount of synthetic sewage feed was prepared within 1 week before use.

Test type:

static

Water media type:

freshwater

Total exposure duration:

3 h

Hardness:

257.2 mg/L CaCO3

Test temperature:

20.6-20.7 degrees Celcius (n=41)

pH:

7.7

Dissolved oxygen:

9.0 mg/L ; 99.0 % saturation

Nominal and measured concentrations:

nominal concentrations (as mg/L BaCL2): 0, 3.2, 10, 32, 100, 320, 1000 mg/L
nominal concentrations (as mg/L test item): 0, 3.8, 11.7, 37.5, 117, 375, 1173 mg/L
measured (nominal of 1000 mg BaCl2/L): t=0: 621.72 mg Ba/L; t=3h: 379.50 mg Ba/L (94% and 58% of nominal, respectively)
(For details see Table 2 in 'Any other information on results incl. tables').

Details on test conditions:

- Storage time of the activated sludge before use: 4 days
- pH-value of the activated sludge: 7.9
- Test vessels: 1000 mL glass beakers without cover
- Amount of test mixture per test vessel: 500 mL
- Volume of microbial inoculum per test mixture: 200 mL
- Volume of synthetic sewage feed per test mixture: 16 mL
- Mixed liquor suspended solids level in the microbial inoculum: 4.00 g/L
- Mixed liquor suspended solids level in the final test mixtures: 1.60 g/L
- Number of test concentrations: 6 plus control
- Number of replicates per test item concentration: 2 plus 1 for chemical analysis
- Number of replicates in the control: 2 plus 1 for chemical analysis
- Aeration of test vessels: 50 to 60 litre of air per hour (ambient air, oil-free air-compressor)

An oximeter OXI 530 (WTW GmbH, D-82362 Weilheim) was used to determine the oxygen content of the test solutions. After three hours of incubation (contact time), the contents of the test vessels were poured into flat-bottom flasks. The oxygen electrode was fitted into the flat-bottom flasks which contained a stirrer bar. The flasks were placed onto a magnetic stirrer. The oximeter was connected to an automated data recording device. The oxygen measurements were taken at intervals of two seconds for up to 10 minutes at the end of the contact period for the content of each test vessel.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

other: BaCl2

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 622 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

element

Remarks:

total Ba

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

>= 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

other: BaCl2

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

>= 622 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

element

Remarks:

total Ba

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Details on results:

Validity criteria: The control respiration rates did not differ more than 15% compared to each other, i.e., 4.0%.

Results with reference substance (positive control):

The EC50 (3 hours) of 3,5- dichlorophenol was in the accepted range of 5 to 30 mg/L, i.e., 9.1 mg/L

Reported statistics and error estimates:

The respiration rate was calculated from the oxygen consumption of the test solutions as mg O2/L·h (also expressed as mg O2/L·h) between 6.5 and 2.5 mg O2/L. The portion of the respiration curve over which the respiration rate was measured was linear. In order to calculate the inhibitory effect of the test item in the test concentration levels, the respiration rate was expressed as a percentage of the mean of the two control respiration rates RC1 and RC2.
Welch-t test for inhomogeneous variances with Bonferroni-Holm adjustment was used to determine the No Observed Effect Concentration (NOEC). Probit Analysis was used to determine the ECx-values. Since in none of the treatments the respiration rate was inhibited statistically significant compared to the controls, the calculation of an ECx-value was not possible.
The statistical software package ToxRat 2.10 Professional (ToxRat Solutions GmbH, Naheweg 15, D-52477 Alsdorf) is used for these calculations.

Table 1: Respiration rates (mg O2/L/h) in the controls and treated vessels: Summary of data

Concentration (mg/L BaCl2)	Replicate	Time (h)	Oxygen consumption (mg/L per hour)	Inhibition (%)
0	1	3	80.22	2.81
3.2	1	3	84.86	-2.81
3.2	2	3	83.33	-0.96
10	1	3	81.52	1.24
10	2	3	81.52	1.24
32	1	3	79.89	3.21
32	2	3	80.00	3.08
100	1	3	78.65	4.71
100	2	3	80.89	2.00
320	1	3	73.54	10.90
320	2	3	76.86	6.88
1000	1	3	73.56	10.88
1000	2	3	74.21	10.09
0	2	3	84.86	-2.81

Table 2: Analytically determined test item concentrations

Test period (h)	Age of test matrix (h)	Nominal concentration Test item (BaCl2·2H2O) [mg/L]	Barium analyzed [mg/L]	Test Item (BaCl2·2H2O) in sample [mg/L]	% of nominal
0	0	0	<LOD	<LOD	-
3	3	0	<LOD	<LOD	-
0	0	1173	621.72	1106.26	94
3	3	1173	379.50	675.27	58

After 3 hours of exposure, 58% of the nominal concentration were measured in the samples of paper-filtered test solution. It is likely that significant portions of Barium were retained in the filter residue (microbial inoculum), which was not analysed. Since the concentrations initially measured in the test solutions were in good agreement with the target concentrations, it can be assumed that the activated sludge was exposed to at least 94% of the nominal concentrations throughout the test.

Validity criteria fulfilled:

yes

Conclusions:

The No Observed Effect Concentration (NOEC) is ≥ 1000 mg BaCl2/L, and hence the Lowest Observed Effect Concentration (LOEC) is > 1000 mg BaCl2/L.

Executive summary:

The effect of barium chloride dihydrate on the respiration rate of bacteria in activated sludge was investigated in a static test system according to the OECD Testing Guideline No. 209 and GLP. The sludge microorganisms were exposed to nominal test item concentrations of 10, 32, 100, 320 and 1000 mg/L and the respiration rate, expressed as oxygen consumption, was measured after an exposure period (contact period) of 3 hours. Inhibition values were calculated by comparing test respiration rates to negative control rates. To determine the actual test item concentrations, samples of the test solution were taken at the start and end of the exposure period and analysed with a validated ICP-OES method.

The validity criteria were fulfulled since the EC50 (3 hours) of 3,5- dichlorophenol was in the accepted range of  5 to 30 mg/L, i.e., 9.1 mg/L and the control respiration rates were within 15% compared to each other.

Since the test item showed no statistically significant inhibitory effect on the respiration rate of the test system at any of the concentrations, no ECx-values were determined. The concentrations initially measured in the test solutions were in good agreement with the target concentrations; therefore it can be assumed that the activated sludge was exposed to at least 94% of the nominal concentrations throughout the test. Thus, the results of this study demonstrate that the test item did not inhibit respiration of activated sludge at a concentration of up to 1000 mg/L. Toxic effect levels (EC20, EC50 and EC80) are therefore greater than 1000 mg BaCl2/L. The No Observed Effect Concentration (NOEC) is ≥ 1000 mg BaCl2/L, and hence the Lowest Observed Effect Concentration (LOEC) is > 1000 mg BaCl2/L.

 

Description of key information

One reliable toxicity data point (Klimisch 1, GLP) has been identified for micro-organisms; a respiration inhibition test with activated sludge . Based on measured total Ba-level in the water column, a  3h-IC50 and 3h-NOEC of >622 and >=622 mg Ba/L, respectively,  was reported by Egeler and Kiefer (2010), using  BaCl2 dihydrated as test substance.

Key value for chemical safety assessment

EC50 for microorganisms:

622 mg/L

EC10 or NOEC for microorganisms:

622 mg/L

Additional information

One reliable toxicity data point (Klimisch 1, GLP) with activated sludge from a (predominantly) domestic sewage plant has been reported by Egeler et al (2010). Inhibition of respiration was the endpoint under consideration. The total fraction of Ba in the test medium was measured. At a nominal concentration of 1000 mg BaCl2/L, the measured Ba-content was 622 mg/L. No inhibition of the respiration rate was noted at this highest concentration level. Therefore - expressed as mg Ba_total/L, the 3h-IC₅₀and 3H-NOEC were >622 and >=622 mg/L, respectively.