Sodium periodate

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

Study period:

12 October 2012 to 2 November 2012

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Definitive Test
Amounts of test material (250, 450, 800, 1400 and 2500 mg) were separately dissolved in water with the aid of ultrasonication and the volumes adjusted to 1 litre to give 250, 450, 800, 1400 and 2500 mg/L stock solutions. An aliquot (200 mL) of the 250 mg/L stock solution was dispersed with synthetic sewage (16 mL), activated sewage sludge (250 mL) and water, to a final volume of 500 mL, to give the required concentration of 100 mg/L. Similarly, aliquots (200 mL) of the 450, 800, 1400 and 2500 mg/L stock solutions were used to prepare the test concentrations of 180, 320, 560 and 1000 mg/L respectively. Five replicate flasks per concentration were prepared. The volumetric flasks containing the stock solutions were inverted several times to ensure homogeneity of the stock solutions.

- Additional Test
Amounts of test material (140 and 250 mg) were separately dissolved in water with the aid of ultrasonication for approximately 10 minutes and the volumes adjusted to 1 litre to give 140 and 250 mg/L stock solutions respectively. An aliquot (200 mL) of the 140 mg/L stock solution was dispersed with synthetic sewage (16 mL), activated sewage sludge (250 mL) and water, to a final volume of 500 mL, to give the required concentration of 56 mg/L. Similarly, an aliquot (200 mL) of the 250 mg/L stock solution was used to prepare the test concentration of 100 mg/L. Five replicate flasks per concentration were prepared. The volumetric flasks containing the stock solutions were inverted several times to ensure homogeneity of the stock solutions.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

INOCULUM
Type: a mixed population of activated sewage sludge micro-organisms.
Source: the aeration stage of the Severn Trent Water PLC sewage treatment plant at Loughborough, Leicestershire, UK which treats predominantly domestic sewage.
Date of collection: 11 October 2012 for the range-finding test, 30 October 2012 for the definitive test and 1 November 2012 for the additional test
Preparation: 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 for the definitive test, the activated sewage sludge (20 litres) was fed synthetic sewage sludge (1000 mL).
On the day of collection for the additional the activated sewage sludge (8 liters) was fed synthetic sewage sludge (400 mL).
A synthetic sewage was added to each test vessel to act as a respiratory substrate, containing the following components dissolved in 1 litre of water with the aid of ultrasonication: 16 g Peptone; 11 g meat extract, 3 g urea, 0.7 g NaCl, 0.4 g CaCl2.2H2O, 0.2 g MgSO4.7H2O and 2.8 g K2HPO4.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test temperature:

20 ± 2 °C

pH:

7.1 - 8.5

Dissolved oxygen:

4.6 - 7.1 mg O2/L (after 30 minutes of contact time in the Definitive Test)
1.6 - 7.9 mg O2/L (after 3 hours of contact time in te Definitive Test)

Nominal and measured concentrations:

- Nominal Test Concentrations (Range-finding Test): 0, 10, 100, 1000 mg/L
- Nominal Test Concentrations (Definitive Test): 100, 180, 320, 560, 1000 mg/L
- Nominal Test Concentrations (Addtional Test): 56, 100 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Conical flask
- Material, size, headspace, fill volume: 500 mL conical flask, filled to 500 mL
- Aeration: 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.
- No. of vessels per concentration (replicates): 5
- No. of vessels per control (replicates): 4
- Suspended solids concentration: 3.0 g/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Deionised reverse osmosis water
- Total organic carbon: less than 1 mg/L

OTHER TEST CONDITIONS
- Photoperiod: Normal laboratory lighting

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : 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 and 2.0 mg O2/L). In the case of 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.

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.

TEST CONCENTRATIONS
- Range finding study
An amount of test material (2500 mg) was dissolved in water with the aid of ultrasonication for approximately 15 minutes and the volume adjusted to 1 litre to give a 2500 mg/L stock solution from which dilutions were made to give 250 and 25 mg/L stock solutions. An aliquot (200 mL) of the 25 mg/L stock solution was dispersed with synthetic sewage (16 mL), activated sewage sludge (250 mL) and water, to a final volume of 500 mL, to give the required concentration of 10 mg/L. Similarly, aliquots (200 mL) of the 250 mg/L and 2500 mg/L stock solutions were used to prepare the test concentrations of 100 and 1000 mg/L. The 1000 mg/L test concentration was prepared in triplicate. The volumetric flasks containing the stock solutions were inverted several times to ensure homogeneity of the stock solutions.
The pH of the test item stock solutions were measured using a WTW pH/Oxi 340I pH and dissolved oxygen meter and adjusted to between pH 7.0 to pH 8.0 if necessary.
The control group was maintained under identical conditions but not exposed to the test material.
- Test concentrations: 1000 mg/L
- Results used to determine the conditions for the definitive study: RANGE-FINDING TEST
The dissolved oxygen concentrations after 30 minutes contact time in a few of the vessels were below 60% of the dissolved oxygen saturation level of 8.9 mg O2/L. 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.
No statistically significant toxic effects were shown at the test concentrations of 10 and 100 mg/L, however statistically significant toxic effects were shown at the test concentration of 1000 mg/L.
Based on these results it was considered necessary to perform a definitive test in order to obtain both a NOEC and an EC50 value for the test material. Based on this information test concentrations of 100, 180, 320, 560 and 1000 mg/L were selected for the definitive test.

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

220 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: 200-240 mg/L (95 % CL)

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

56 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Details on results:

DEFINITIVE TEST
Statistically significant toxic effects were shown at all the test concentrations employed. Therefore based on these results it was considered necessary to perform an additional test employing the test concentrations of 56 and 100 mg/L in order to obtain the NOEC.
The coefficient of variation of oxygen uptake in the control vessels was 6.9 % and the specific respiration rate of the controls was 25.55 mg oxygen per gram dry weight of sludge per hour. The validation criteria were therefore 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.
The dissolved oxygen concentrations after 30 minutes contact time in a few vessels were below 60 % of the dissolved oxygen saturation level of 8.9 mg O2/L. 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.

ADDITIONAL TEST
No statistically significantly toxic effects were shown at the test concentration of 56 mg/L, however statistically significantly toxic effects were shown at the test concentration of 100 mg/L. Based on these results the No Observed Effect Concentration (NOEC) after 3 hours exposure was 56 mg/L.
The coefficient of variation of oxygen uptake in the control vessels was 3.8 % and the specific respiration rate of the controls was 33.28 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.
The dissolved oxygen concentrations after 30 minutes contact time in most of vessels were below 60 % of the dissolved oxygen saturation level of 8.9 mg O2/L. 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.

Results with reference substance (positive control):

A reference item, 3,5-dichlorophenol, was included in the range-finding, definitive, and additional test at concentrations of 3.2, 10 and 32 mg/L in order to confirm the suitability of the inoculum. A stock solution of 0.5 g/L was prepared by dissolving the reference item directly in water with the aid of ultrasonication for approximately 30 minutes. The pH of this stock solution was measured to be pH 6.8 and was adjusted to pH 7.1 using 1.0 M NaOH. Aliquots (3.2, 10 and 32 mL) of the stock solution were removed and dispersed with activated sewage sludge (250 mL), synthetic sewage (16 mL) and water to give the final concentrations of 3.2, 10 and 32 mg/L. The volumetric flask containing the reference item was inverted several times to ensure homogeneity of the solution.

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

The following results were derived:
EC20: 3.1 mg/L
EC50: 9.3 mg/L (95 % confidence limits 7.6 - 11 mg/L)
EC80: 27 mg/L

The validation criteria for the reference material were satisfied (i.e. EC50 (3 hour contact time) within the range 2 to 25 mg/L).

Reported statistics and error estimates:

The respiration rate, R, expressed in mg O2/L/h, was calculated from the linear part of the recorded oxygen decrease graph according to the following equation:
R = [(Q1 - Q2) /Δt] x 60
Where
Q1 = the oxygen concentration at the beginning of the selected section of the linear phase (mg/L).
Q2 = the oxygen concentration at the end of the selected section of the linear phase (mg/L).
Δt = the time interval between the beginning and end of the selected section of the linear phase (min).
 
The specific respiration rate, Rs, expressed as mg O2/g/h (the amount of oxygen consumed per gram dry weight of sludge per hour) was deduced according to the following equation:
Rs = R / SS
Where
SS = the concentration of suspended solids in the test mixture (g/L).
 
The percentage inhibition was calculated according to the following equation:
% inhibition = [1 - (R / Rbc)] x 100
Where
Rbc = the mean respiration rate of the blank controls.
 
The percentage inhibition values were plotted against concentration for the reference material only; a line fitted using the Xlfit software package (IDBS) and the EC20, EC50 and EC80 values determined from the equation for the fitted line.
 
95 % confidence limits were calculated for the reference material EC50 value using the method of Litchfield and Wilcoxon (1949).
 
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 of Contact Time in the Range-Finding Test

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
	R1	4.7	4	1.9	42.00	-
Control	R2	4.7	4	2.1	39.00	-
	R3	6.0	6	2.0	40.00	-
	R4	6.5	7	2.2	36.85	-
	10	3.2	2	1.8	42.00	[6]
Test	100	5.2	5	2.2	36.00	9
Material	1000 R1	8.4	10	8.0	2.40	94
	1000 R2	8.7	10	8.2	3.00	92
	1000 R3	8.4	10	8.0	2.40	94
Reference	3.2	5.8	6	2.2	36.00	9
Material	10	7.0	10	3.5	21.00	47
	32	7.9	10	6.4	9.00	77

[increase in respiration rate as compared to controls]

R1 - R4 = replicates 1 to 4

Table 2: Oxygen Consumption Rates and Percentage Inhibition Values after 3 hours of Contact Time in the Definitive Test

Nominal conc. (mg/L)	Initial O2 reading (mg O2/L)	Measurement period (min.)	Final O2 reading (mg O2/L)	O2 consumption rate (mg O2/L)	% inhibition
Control R1	3.1	2	1.7	42.00	-
R2	3.5	3	1.6	38.00	-
R3	3.7	3	1.8	38.00	-
R4	4.0	3	2.1	38.00	-
R5	3.6	3	1.6	40.00	-
R6	5.5	6	2.1	34.00	-
Test material       100 R1	3.3	2	2.1	36.00	6
100 R2	5.7	7	2.3	29.14	24
100 R3	5.9	7	2.3	30.86	19
100 R4	4.1	3	2.3	36.00	6
100 R5	4.5	4	1.9	39.00	[2]
180 R1	6.0	9	2.3	24.67	36
180 R2	5.9	9	2.4	23.33	39
180 R3	5.3	6	2.4	29.00	24
180 R4	5.5	9	1.8	24.67	36
180 R5	5.8	8	2.4	25.50	33
320 R1	7.2	10	5.9	7.80	80
320 R2	7.3	10	5.9	8.40	78
320 R3	6.8	6	5.9	9.00	77
320 R4	7.0	10	5.5	9.00	77
320 R5	7.3	10	5.8	9.00	77
560 R1	7.8	10	7.2	3.60	91
560 R2	7.7	8	7.3	3.00	92
560 R3	7.8	10	7.3	3.00	92
560 R4	7.8	10	7.2	3.60	91
560 R5	7.8	10	7.3	3.00	92
1000 R1	7.9	10	7.6	1.80	95
1000 R2	7.8	10	7.6	1.20	97
1000 R3	7.8	7	7.7	0.86	98
1000 R4	7.8	10	7.6	1.20	97
1000 R5	7.9	10	7.8	0.86	98
3,5-dichlorophenol    3.2	5.4	7	2.0	34.00	11
10	6.6	6	3.1	21.00	45
32	7.9	10	6.4	9.00	77

[increase in respiration rate compared to controls]

R1 – R6 = Replicates 1 to 6

Table 3: Oxygen Consumption Rates and Percentage Inhibition Values after 3 hours of Contact Time in the Additional Test

Nominal conc. (mg/L)	Initial O2 reading (mg O2/L)	Measurement period (min)	Final O2 reading (mg O2/L)	O2 consumption rate (mg O2/L)	% inhibition
Control R1	4.8	3	2.5	46.00	-
R2	5.0	4	2.0	45.00	-
R3	4.7	3	2.2	50.00	-
R4	5.1	4	2.0	46.50	-
R5	4.8	4	1.6	48.00	-
R6	4.6	3	2.3	46.00	-
Test material 56 R1	4.8	4	1.8	45.00	4
56 R2	4.4	3	2.2	44.00	6
56 R3	4.2	3	2.0	44.00	6
56 R4	4.2	3	1.8	48.00	[2]
56 R5	4.7	4	1.6	46.50	1
100 R1	5.2	4	2.3	43.50	7
100 R2	5.2	5	1.8	40.80	13
100 R3	5.3	4	2.3	45.00	4
100 R4	5.1	4	2.2	43.50	7
100 R5	5.5	5	2.0	42.00	10
3,5-dichlorophenol 3.2	4.5	3	2.6	38.00	19
10	7.0	10	3.5	21.00	55
32	8.1	10	6.8	7.80	83

[increase in respiration rate compared to controls]

R1 – R6 = Replicates 1 to 6

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the study, the 3-hour EC50 for the test material was determined to be 220 mg/L while the 3-hour No Observed Effect Concentration (NOEC) was determined to be 56 mg/L.

Executive summary:

The potential of the test material to cause toxic effects to microorganisms was investigated in a GLP study which was conducted in accordance with the standardised guideline OECD 209.

Following a preliminary range-finding test, a definitive test was employed in order to try to obtain the No Observed Effect Concentration (NOEC) and an EC50 value for the test material. Activated sewage sludge was exposed to an aqueous solution of the test material at concentrations of 100, 180, 320, 560 and 1000 mg/L (5 replicates for each test 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.

Unfortunately the No Observed Effect (NOEC) was not obtained, therefore an additional test was performed for a period of 3 hours at a temperature of 20 ± 2 °C with the addition of a synthetic sewage as a respiratory substrate employing the test concentrations of 56 and 100 mg/L (5 replicates for each test concentration) in order to obtain the NOEC.

The rate of respiration was determined after 3 hours contact time and compared to data for the control and a reference item, 3,5-dichlorophenol.

The effect of the test material on the respiration of activated sewage sludge gave a 3-hour EC50 value of 220 mg/L, 95 % confidence limits 200 - 400 mg/L.

The reference item gave a 3-hour EC50 value of 12 mg/L, 95 % confidence limits 9.5 - 15 mg/L.

In the additional test the No Observed Effect Concentration (NOEC) after 3 hours exposure was 56 mg/L. The reference item gave a 3-hour EC50 value of 9.3 mg/L, 95 % confidence limits 7.6 - 11 mg/L.

Description of key information

The 3-hour EC50 for the test material to microorganisms was 220 mg/L with 95 % confidence limits of 200 - 400 mg/L.  

Key value for chemical safety assessment

EC50 for microorganisms:

220 mg/L

EC10 or NOEC for microorganisms:

56 mg/L

Additional information

The potential of the test material to cause toxic effects to microorganisms was investigated in a GLP study which was conducted in accordance with the standardised guideline OECD 209. The study was assigned a reliability score of 1 in accordance with the principles for assessing data quality as set forth by Klimisch et al (1997).

Following a preliminary range-finding test, a definitive test was employed in order to try to obtain the No Observed Effect Concentration (NOEC) and an EC50 value for the test material. Activated sewage sludge was exposed to an aqueous solution of the test material at concentrations of 100, 180, 320, 560 and 1000 mg/L (5 replicates for each test 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.

Unfortunately the No Observed Effect (NOEC) was not obtained, therefore an additional test was performed for a period of 3 hours at a temperature of 20 ± 2 °C with the addition of a synthetic sewage as a respiratory substrate employing the test concentrations of 56 and 100 mg/L (5 replicates for each test concentration) in order to obtain the NOEC.

The rate of respiration was determined after 3 hours contact time and compared to data for the control and a reference item, 3,5-dichlorophenol.

The effect of the test material on the respiration of activated sewage sludge gave a 3-hour EC50 value of 220 mg/L, 95 % confidence limits 200 - 400 mg/L.

The reference item gave a 3-hour EC50 value of 12 mg/L, 95 % confidence limits 9.5 - 15 mg/L.

In the additional test the No Observed Effect Concentration (NOEC) after 3 hours exposure was 56 mg/L. The reference item gave a 3-hour EC50 value of 9.3 mg/L, 95 % confidence limits 7.6 - 11 mg/L.