Dihydrogen hexahydroxyplatinate

Toxicity to microorganisms

Administrative data

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:

other: GLP-compliant, guideline study, available as an unpublished report, no restrictions, fully adequate for assessment.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

yes

Remarks:

The T°C in the 1st and 2nd experiment was slightly higher, the measurement period was partly less than 5 min, and in some cases the O2 concentration within the measurement period was not between 6.5-2.5 mg/L.

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)

Deviations:

yes

Remarks:

The T°C in the 1st and 2nd experiment was slightly higher, the measurement period was partly less than 5 min, and in some cases the O2 concentration within the measurement period was not between 6.5-2.5 mg/L.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Dihydrogen hexahydroxyplatinate

EC Number:

257-471-2

EC Name:

Dihydrogen hexahydroxyplatinate

Cas Number:

51850-20-5

Molecular formula:

H6O6Pt.2H

IUPAC Name:

hexahydroxyplatinate(2-)

Test material form:

solid: crystalline

Sampling and analysis

Analytical monitoring:

no

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: treatments were prepared by putting the appropriate amount of test item (1st experiment 0.5, 5.1, 50.6 and 500.1 mg test item, 2nd experiment 500 mg test item, and 3rd experiment 50 and 160 mg test item) into the respective test vessel, adding 16 mL nutrient solution and then water to give 250 mL. Additionally, the second series in the 1st and 2nd experiments with ATU was prepared by putting additional 2.5 mL ATU stock solution into the respective test vessel. Then, 250 mL inoculum was added in five minutes intervals and the mixture was aerated.
- Controls: activated sludge containing nutrient solution and tap water without test item

Test organisms

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Name and location of sewage treatment plant where inoculum was collected: the sludge was taken from the activation basin of the ESN (Stadtentsorgung Neustadt) sewage treatment plant D-67435 NW-Lachen-Speyerdorf.
- Preparation of inoculum for exposure: the sludge was filtrated, washed with tap water and resuspended in tap water. Volume was adapted to the desired content of dry matter. The activated sludge was aerated until usage in the test and fed daily with 50 mL/L synthetic sewage feed.
- Initial biomass concentration: not reported

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Remarks on exposure duration:

Experiment 2

Post exposure observation period:

Based on the results of experiment 1, experiment 2 was conducted as a limit test. Because statistically significant inhibition was observed for the test item in the test series without ATU, a third experiment was performed for determination of the NOEC with two concentrations of the test item and without ATU.

Test conditions

Hardness:

0.9 mmol/L

Test temperature:

1st experiment: 21.3 – 24.0 °C
2nd experiment: 22.2 – 24.2 °C
3rd experiment: 20.4 – 21.9 °C

pH:

1st experiment: 8.1 (with ATU) and 8.0-8.1 (without ATU) at the end of the test
2nd experiment: 7.8 - 7.9 (with ATU) and 7.7 - 8.0 (without ATU) at the end of the test
3rd experiment: 7.5 - 7.7 (without ATU) at the end of the test

Nominal and measured concentrations:

Nominal concentrations - 1st experiment: 1, 10, 100 and 100 mg test item/L
Nominal concentrations - 2nd experiment: 1000 mg test item/L
Nominal concentrations - 3rd experiment: 100 and 320 mg test item/L

Details on test conditions:

TEST SYSTEM
- Test vessel: glass beakers, containing 500 mL test solution, as test vessels. Narrow-neck glass bottles with flat bottoms as measuring flasks
- Aeration: purified air using Pasteur pipettes, flow rate of approximately 0.75L/min
- No. of vessels per concentration (replicates) - 1st experiment: 1 (with and without ATU)
- No. of vessels per concentration (replicates) - 2nd experiment: 5 (with and without ATU)
- No. of vessels per concentration (replicates) - 3rd experiment: 5 (without ATU)
- No. of vessels per control (replicates): 6 (with and without ATU)
- Sludge concentration (weight of dry solids per volume): 2.22 g suspensed solids/L (1st exp.), 3.20 g suspended solids/L (2nd exp.), 2.76 g suspended solids/L (3rd exp.)
- Weight of dry solids per volume in the test: 1.11 g suspended solids/L (1st exp.), 1.60 g suspended solids/L (2nd exp.), 1.38 g suspended solids/L (3rd exp.)
- Nutrients provided for bacteria: 16 mL per vessel of synthetic sewage, containing peptone 16.0 g, meat extract 11.0 g, urea 3.0 g, NaCl 0.7 g, CaCl2.2H2O 0.4 g, MgSO4.7H2O 0.2 g, K2HPO4 2.8 g and deionised water ad 1000 mL. The pH of solution was 7.0.
- Nitrification inhibitor used: N-allylthiourea (ATU)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: tap water

EFFECT PARAMETERS MEASURED: the respiration rate was measured after three hours of contact time by measurement of the oxygen concentration over a period of maximum five minutes.

TEST CONCENTRATIONS
- Range finding study (1st exp.)
- Test concentrations: 0, 10, 101, 1000 mg test item/L
- Results used to determine the conditions for the definitive study: no significant inhibition was observed in the highest test concentration

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Results and discussion

Effect concentrations

Effect concentrations 1

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

Effect concentrations 2

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 648 mg/L

Nominal / measured:

nominal

Conc. based on:

element

Remarks:

Pt

Basis for effect:

inhibition of total respiration

Effect concentrations 3

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

320 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Effect concentrations 4

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

207 mg/L

Nominal / measured:

nominal

Conc. based on:

element

Remarks:

Pt

Basis for effect:

inhibition of total respiration

Effect concentrations 5

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Effect concentrations 6

Key result

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

648 mg/L

Nominal / measured:

nominal

Conc. based on:

element

Remarks:

Pt

Basis for effect:

inhibition of total respiration

Effect concentrations 7

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 heterotrophic respiration

Effect concentrations 8

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 648 mg/L

Nominal / measured:

nominal

Conc. based on:

element

Remarks:

Pt

Basis for effect:

inhibition of heterotrophic respiration

Effect concentrations 9

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 heterotrophic respiration

Effect concentrations 10

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

648 mg/L

Nominal / measured:

nominal

Conc. based on:

element

Remarks:

Pt

Basis for effect:

inhibition of heterotrophic respiration

Effect concentrations 11

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of heterotrophic respiration

Effect concentrations 12

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

> 648 mg/L

Nominal / measured:

nominal

Conc. based on:

element

Remarks:

Pt

Basis for effect:

inhibition of heterotrophic respiration

Details on results:

3h EC10 (respiration rate) = 1000 mg/L (nominal based on test mat.)
3h EC10 (respiration rate) = 648 mg/L (nominal based on element)
3h EC10 (heterotrophic respiration) > 1000 mg/L (nominal based on test mat.)
3h EC10 (heterotrophic respiration) > 648 mg/L (nominal based on element)

Results with reference substance (positive control):

Without ATU - 1st experiment: 3-hour EC50 15 mg/L (95% CL 9.0 - 23 mg/L); 2nd experiment: 3-hour EC50 14 mg/L (95% CL 11 - 17 mg/L); 3rd experiment: 3-hour EC50 8.4 mg/L (95% CL 5.3 - 12 mg/L) all of which are within the recommended range of 2- 25 mg/L.
With ATU - 1st experiment: 3-hour EC50 15 mg/L (95% CL 8.9 - 28 mg/L) and 2nd experiment: 3-hour EC50 17 mg/L (95% CL <26 mg/L) both are within the recommended range of 5- 40 mg/L.

Reported statistics and error estimates:

Oxygen consumption was calculated using linear regression.
EC50 was calculated using linear fit on a probability-logarithmic scale using Origin™ software.
NOEC was determined by testing equality of variance and then using either a t-test or WEIR test, in order to evaluate statistically significant differences between treatment groups and control groups.

Any other information on results incl. tables

Table 1. First experiment (range-finding test) - Oxygen consumption and % Inhibition after 3 hours of exposure of activated sludge to dihydrogen hexahydroxyplatinate with N-allylthiourea (ATU)

Content	Concentration (mg test item/L)	O2 consumption in (mg/(L*h))	% Inhibition
Control	0	35.748	-
	0	35.199	-
	0	34.813	-
	0	35.575	-
	0	32.817	-
	0	32.998	-
Test item	1	32.520	5.8
	10	32.072	7.1
	101	35.365	-2.4
	1000	32.318	6.4
Positive control	Concentration (mg positive control/L)
	5	32.258	6.6
	10	22.986	33.4
	20	9.515	72.4
	40	5.642	83.7

Table 2. First experiment (range-finding test) - Oxygen consumption and % Inhibition after 3 hours of exposure of activated sludge to dihydrogen hexahydroxyplatinate without ATU

Content	Concentration (mg test item/L)	O2 consumption in (mg/(L*h))	% Inhibition
Control	0	35.491	-
	0	35.142	-
	0	34.177	-
	0	34.714	-
	0	35.315	-
	0	35.938	-
Test item	1	34.583	1.6
	10	34.093	3.0
	101	34.700	1.2
	1000	34.297	2.4
Positive control	Concentration (mg positive control/L)
	5	32.021	8.9
	10	21.794	38.0
	20	10.016	71.5
	40	5.3940	84.6

Table 3. Second experiment (limit test) - Oxygen consumption and % Inhibition after 3 hours of exposure of activated sludge to dihydrogen hexahydroxyplatinate with ATU

Content	Concentration (mg test item/L)	O2 consumption in (mg/(L*h))	% Inhibition
Control	0	67.698	-
	0	67.814	-
	0	70.990	-
	0	64.839	-
	0	60.654	-
	0	63.872	-
Test item	1002	64.689	2.0
	1000	59.562	9.7
	1001	67.510	-2.3
	1000	58.686	11.1
	1001	57.221	13.3
Positive Control	Concentration (mg positive control/L)
	5	67.235	-1.9
	10	47.370	28.2
	20	27.519	58.3
	40	12.583	80.5

Table 4. Second experiment (limit test) - Oxygen consumption and % Inhibition after 3 hours of exposure of activated sludge to dihydrogen hexahydroxyplatinate without ATU

Content	Concentration (mg test item/L)	O2 consumption in (mg/(L*h))	% Inhibition
Control	0	75.581	-
	0	71.959	-
	0	72.532	-
	0	73.974	-
	0	78.714	-
	0	73.554	-
Test item	1000	67.902	8.7
	1000	66.139	11.1
	1000	65.534	11.9
	1002	67.229	9.6
	1000	66.609	10.5
Positive Control	Concentration (mg positive control/L)
	5	62.066	16.6
	10	44.055	40.8
	20	26.079	64.9
	40	13.802	81.4

Table 5. Third experiment - Oxygen consumption and % Inhibition after 3 hours of exposure of activated sludge to dihydrogen hexahydroxyplatinate without ATU

Content	Concentration (mg test item/L)	O2 consumption in (mg/(L*h))	% Inhibition
Control	0	49.368	-
	0	48.389	-
	0	50.986	-
	0	49.711	-
	0	51.952	-
	0	51.600	-
Test item	100	53.089	-5.5
	100	51.434	-2.2
	101	52.379	-4.1
	100	51.553	-2.4
	100	53.548	-6.4
	320	49.985	0.7
	321	53.109	-5.5
	321	48.301	4.0
	319	49.747	1.2
	321	51.081	-1.5
Positive control	Concentration (mg positive control/L)
	5	35.510	29.5
	10	20.831	58.6
	20	9.869	80.4
	40	5.074	89.9

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

As regards the inhibition of the respiration of activated sludge when exposed to dihydrogen hexahydroxyplatinate, the 3-h EC50, EC10 and NOEC without ATU were determined to be > 1000 mg test item/L (> 648 mg Pt/L), 1000 mg test item/L (648 mg Pt/L), and 320 mg test item/L (207 mg Pt/L), respectively. The 3-h EC50 and EC10 with ATU were both determined to be > 1000 mg test item/L (> 648 mg Pt/L), and the relative NOEC was determined to be 1000 mg test item/L (648 mg Pt/L).

Executive summary:

An activated sludge respiration inhibition test was conducted with dihydrogen hexahydroxyplatinate. The study is reliable without restriction, being GLP-compliant and having followed the standard test guidelines (OECD 209 and EU Method C.11). Three experiments were performed, including a range-finding test, a limit test using the concentration of 1000 mg/L, and a third test using the concentrations of 100 and 320 mg/L. Two test series were tested with and without the nitrification inhibitor N- allylthiourea (ATU) to discern between inhibition of nitrificators and inhibition of the total population. A positive control was used, and all validity criteria were met.

The 3-h EC50, EC10 and NOEC without ATU were determined to be > 1000 mg test item/L (> 648 mg Pt/L), 1000 mg test item/L (648 mg Pt/L), and 320 mg test item/L (207 mg Pt/L), respectively. The 3-h EC50 and EC10 with ATU were both determined to be > 1000 mg test item/L(> 648 mg Pt/L), and the relative NOEC was determined to be 1000 mg test item/L (648 mg Pt/L).