Dipraseodymium trioxide

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 July 2012 to 20 July 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Read-across performed with structurally similar substance.

Reason / purpose for cross-reference:

other: read-across target

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

yes

Remarks:

see below

Principles of method if other than guideline:

In some instances, the initial and final dissolved oxygen concentrations were outside those recommended in the test guidelines (7.0 mg O₂/L and 2.0 mg O₂/L respectively). This was considered to have had no adverse effect on the results of the study given that in all cases the oxygen consumption rate was determined over the linear portion of the oxygen consumption trace.

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Test water: The water used for the test was deionised reverse osmosis water containing less than 1 mg/L Dissolved Organic Carbon.
- Method: 5, 50 and 500 (in triplicate) mg of test material were each separately dispersed in approximately 200 mL of deionised reverse osmosis water and subjected to ultrasonication for approximately 15 minutes and stirred with a magnetic stirrer for 24 hours. All test vessels were shielded from the light during mixing. 16 mL of synthetic sewage, 250 mL of activated sewage sludge and water were added to a final volume of 500 mL to give the required nominal concentrations of 10, 100 and 1000 mg/L.
- Controls: The positive control 3,5-dichlorophenol was prepared as a 0.5 g/L stock solution by dissolving directly into water with the aid of ultrasonication (30 minutes). The pH of this stock solution was measured to be 6.2 and was adjusted to pH 7.5 using 1.0 M NaOH. Aliquots (3.2, 10 and 32 mL) of the stock solution were removed and dispersed with activated sludge (250 mL), synthetic sewage (16 mL) and water to give the final concentrations of 3.2, 10 and 32 mg/L.
A negative control was also included.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

A mixed population of activated sewage sludge micro-organisms was obtained from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK which treats predominantly domestic sewage.

- Preparation of inoculum for exposure: The activated sewage sludge sample was maintained on continuous aeration in the laboratory at a temperature of approximately 21 °C overnight prior to use in the test. On the day of collection the activated sewage sludge (5 litres) was fed synthetic sewage sludge (250 mL).
- Suspended solids: 3.0 g/L

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Post exposure observation period:

None

Hardness:

NDA

Test temperature:

20 ± 2 °C

pH:

7.0 - 7.3 (after 0 hours exposure); 7.4 - 7.8 (after 3 hours exposure)

Dissolved oxygen:

Initial reading: 3.8 - 8.9 mg/L, reading after 30 minutes contact time: 3.2 - 9.8 mg/L and Final reading: 1.5 - 8.1 mg/L

Salinity:

N/A

Nominal and measured concentrations:

Nominal test concentrations of 10, 100 and 1000 mg/L.

Details on test conditions:

SYNTHETIC SEWAGE
A synthetic sewage of the following composition was added to each test vessel to act as a respiratory substrate:
16 g: Peptone
11 g: Meat extract
3 g: Urea
0.7 g: NaCl
0.4 g: CaCl2.2H2O
0.2 g: MgSO4.7H2O
2.8 g: K2HPO4
dissolved in 1 litre of water with the aid of ultrasonication.

The pH of the synthetic sewage stock used to feed the activated sewage sludge and used for the study was 7.1. The pH value was measured using a WTW pH/Oxi 340I pH and dissolved oxygen meter.

PREPARATION OF TEST SYSTEM
At time "0" 16 mL of synthetic sewage was diluted to 250 mL with water and 250 mL of inoculum added in a 500 mL conical flask (first control). The mixture was aerated with clean, oil-free compressed air via narrow bore glass tubes at a rate of approximately 0.5 - 1 litre per minute. Thereafter, at 15 minute intervals the procedure was repeated for the second control followed by the reference material vessels with appropriate amounts of the reference material added. The test material vessels were prepared and two further control vessels were prepared.

As each vessel reached 3 hours contact time, an aliquot was removed from the conical flask and poured into the measuring vessel (250 mL darkened glass Biological Oxygen Demand (BOD) bottle) and the rate of respiration measured using a Yellow Springs dissolved oxygen meter fitted with a BOD probe. The contents of the measuring vessel were stirred constantly by magnetic stirrer. The rate of respiration for each flask was measured over the linear portion of the oxygen consumption trace (where possible between approximately 7.0 mg/L and 2.0 mg/L). In the case of a rapid oxygen consumption, measurements may have been outside this range but the oxygen consumption was always within the linear portion of the respiration curve. In the case of low oxygen consumption, the rate was determined over an approximate 10 minute period.

OTHER TEST CONDITIONS
The test was conducted under normal laboratory lighting. The test material was dispersed directly in water. The control group was maintained under identical conditions but not exposed to the test material.

EFFECT PARAMETERS MEASURED
Observations were made on the test preparations throughout the test period. Observations of the test material vessels at 0 hours were made prior to addition of activated sewage sludge. The pH of the control, reference and test material preparations was measured at 0 hours and prior to measurement of the oxygen consumption rate after 3 hours contact time. The oxygen concentrations in all vessels were measured after 30 minutes contact time.

- Validity of the test:
The results of the study are considered valid if
(i) the EC50 (3 hour contact time) for 3,5-dichlorophenol lies within the range 2 to 25 mg/L
(ii) the specific respiration rate of the blank controls should not be less than 20 mg oxygen per gram dry weight of sludge per hour
(iii) the coefficient of variation of oxygen uptake rate in control replicates should not be more than 30 % at the end of the test.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Details on results:

No statistically significant toxic effects were shown at any of the test concentrations employed. It was therefore considered justifiable not to perform a definitive test.
It was considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg/L.

Oxygen consumption rates and percentage inhibition values for the control, test and reference materials are given in Table 1.

The dissolved oxygen concentrations after 30 minutes contact time in all vessels were above 60 to 70 % of the dissolved oxygen saturation level of 8.9 mg O₂/L with the exception of the 100 mg/L test material replicate, 1000 mg/L test item replicates R1, R2 and R3 and controls R3 and R4 vessels showing between 36 to 48% saturation. This deviation was considered to have had no adverse effect on the study given that all oxygen consumption values were measured/calculated over the linear portion of the traces.

The coefficient of variation of oxygen uptake in the control vessels was 10 % and the specific respiration rate of the controls was 32.80 mg oxygen per gram dry weight of sludge per hour. The validation criteria have therefore been satisfied.

In some instances, the initial and final dissolved oxygen concentrations were outside those recommended in the test guidelines (7.0 mg O₂/L and 2.0 mg O₂/L respectively). This was considered to have had no adverse effect on the results of the study given that in all cases the oxygen consumption rate was determined over the linear portion of the oxygen consumption trace.

Visible particles of the test item were visible on the sides of the flask at all test substance concentrations and at all time points.

Results with reference substance (positive control):

The following results were derived:

3 h EC20 2.6 mg/L
3 h EC50 7.6 mg/L (95 % Confidence Limits 6.0 - 9.7 mg/L)
3 h EC80 22.2 mg/L

The validation criterion for the reference item EC50 value was satisfied.

Reported statistics and error estimates:

One way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett, 1955) was carried out on the oxygen consumption data after 3 hours for the control and all test concentrations to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS, 1999 - 2001).

Table 1: Oxygen Consumption Rates and Percentage Inhibition Values after 3 Hours Contact Time

Nominal Concentration (mg/L)		Initial O₂ Reading (mg O₂/L)	Measurement Period (minutes)	Final O₂ Reading (mg O₂/L)	O₂ Consumption Rates (mg O₂/L/hour)	% Inhibition
Control	R1	3.8	2	2.1	51.00	-
	R2	5.1	5	1.6	42.00	-
	R3	5.9	5	1.5	52.80	-
	R4	5.8	4	2.4	51.00	-
Test Material	10	6.1	5	1.8	51.60	[5]
	100	6.1	5	1.9	50.40	[2]
	1000 R1	6.2	5	2.0	50.40	[2]
	1000 R2	6.2	5	1.9	51.60	[5]
	1000 R3	6.6	6	2.0	46.00	7
Reference Material	3.2	6.2	7	1.9	36.86	25
	10	8.0	10	4.7	19.80	60
	32	8.9	9	8.1	5.33	89

[ ] = Increase in respiration rate as compared to the controls

R1 - R4 = Replicates 1 to 4

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the study, the effect of the test material on the respiration of activated sewage sludge micro-organisms gave a 3 hour EC50 value of greater than 1000 mg/L. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 1000 mg/L.

Executive summary:

The effect of the test material on the respiration of activated sewage sludge was investigated in accordance with the standardised guideline OECD 209.

During the study activated sewage sludge was exposed to an aqueous dispersion of the test material at concentrations of 10, 100 and 1000 mg/L (3 replicates of the 1000 mg/L concentration) for a period of 3 hours at a temperature of 20 ± 2°C with the addition of a synthetic sewage as a respiratory substrate. The rate of respiration was determined after 3 hours contact time and compared to data for the control and a reference material, 3,5-dichlorophenol.

Under the conditions of the test the 3 hour EC50 was determined to be in excess of 1000 mg/L and the NOEC was determined to be 1000 mg/L. It was considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg/L. The reference material gave a 3 hour EC50 value of 7.6 mg/L (95 % confidence limits: 6.0 - 9.7 mg/L).

Reference 2

Endpoint:

activated sludge respiration inhibition testing

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study conducted on read-across material

Justification for type of information:

Read-across performed with structurally similar substance.

Reason / purpose for cross-reference:

read-across source

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 1 000

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Description of key information

Read-across performed with structurally similar substance.

Under the conditions of the test the 3 hour EC50 was determined to be in excess of 1000 mg/L and the NOEC was determined to be 1000 mg/L. It was considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg/L. The reference material gave a 3 hour EC50 value of 7.6 mg/L (95 % confidence limits: 6.0 - 9.7 mg/L).

Key value for chemical safety assessment

EC50 for microorganisms:

1 000 mg/L

Additional information

Read-across performed with structurally similar substance.

The effect of the test material on the respiration of activated sewage sludge was investigated in accordance with the standardised guideline OECD 209. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

During the study activated sewage sludge was exposed to an aqueous dispersion of the test material at concentrations of 10, 100 and 1000 mg/L (3 replicates of the 1000 mg/L concentration) for a period of 3 hours at a temperature of 20 ± 2 °C with the addition of a synthetic sewage as a respiratory substrate. The rate of respiration was determined after 3 hours contact time and compared to data for the control and a reference material, 3,5-dichlorophenol.

Under the conditions of the test the 3 hour EC50 was determined to be in excess of 1000 mg/L and the NOEC was determined to be 1000 mg/L. It was considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg/L. The reference material gave a 3 hour EC50 value of 7.6 mg/L (95 % confidence limits: 6.0 - 9.7 mg/L).