tetraammine palladium (II) hydrogen carbonate

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:

10 to 14 March 1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant, guideline study, available as unpublished report.

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:

The rate of respiration was measured over approximately a 10 minute period after 30 minutes and 3 hours contact time.

Vehicle:

no

Details on test solutions:

400 mg of test material were dissolved in water with the aid of ultrasonic disruption and the volume adjusted to 1 l to give a 400 mg/l stock solution.
Aliquots (12.5, 22.5, 40, 70 and 125 ml) of this stock solution were removed and dispersed with activated sewage sludge, standard synthetic sewage and water to give the final concentrations of 10, 18, 32, 56 ans 100 mg/l.
The test water was laboratory tap water dechlorinated by passage through an activated carbon filter.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Laboratory culture: A mixed population of activated sewage sludge micro-organisms was obtained on 23 March 1995 from the aeration stage of the Severn Trent Water Pic sewage treatment plant at Belper, Derbyshire, U . K .
- Preparation of inoculum for exposure: The activated sewage sludge sample was maintained on continuous aeration in the laboratory at a temperature of 21°C and was used on the day of collection. The pH of the sample was 6.8 and the suspended solids equal to 4.0 g/l prior to use.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Hardness:

100 mg/l as CaCO3

Test temperature:

21 °C

pH:

6.8

Nominal and measured concentrations:

Only nominal concentrations were used

Details on test conditions:

TEST SYSTEM
- Test vessel: conical flasks
- Material, size, headspace, fill volume: 500 ml (16 ml of synthetic sewage diluted to 300 ml with water and 200 ml of inoculum)
- Aeration: aerated with compressed air via narrow bore glass tubes at approximately 0.5-1.0 litre per minute
- Renewal rate of test solution (frequency/flow rate): no renewal
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 2

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: laboratory tap water dechlorinated by passage through an activated carbon filter
- Culture medium different from test medium: standard synthetic sewage

OTHER TEST CONDITIONS
- Photoperiod: normal laboratory lighting

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Rate of respiration measured after 30 minutes and 3 hours contact time

TEST CONCENTRATIONS
A range-finding study was initially performed at test concentrations of 1.0, 10, 100 and 1000 mg/l. Significant inhibition of respiration was observed at 100 and 1000 mg/l, however, there were no significant effects on respiration at 1 and 10 mg/l. Based on this information, test concentrations of 10, 18, 32, 56 and 100 mg/l were selected for the definitive study.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Duration:

30 min

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

35 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Details on results:

In order to calculate the inhibitory effect of test and reference materials, the respiration rate was expressed as a percentage of the two control respiration rates:
% inhibition = { 1 – [RS/(RC1 + RC2)]} *100
Where RS is the oxygen consumption rate for test or reference sample and RC1 and RC2 are the oxygen consumption rates for controls 1 and 2. Percentage inhibition was plotted against concentration and the EC50 values derived by inspection of the graph.

Results with reference substance (positive control):

The 3,5-dichlorophenol 30minute-EC50 and 3hour- EC50 were 16 and 11 mg/l respectively. The variation in respiration rate for the 30 minutes and 3 hour EC50 were ±1% and ±3% respectively.

Validity criteria fulfilled:

yes

Conclusions:

in a OECD- and GLP-compliant study, a 3 hour-EC50 of 35 mg/l for respiratory inhibition was determined for tetraamminepalladium(II) hydrogen carbonate.

Executive summary:

Tetramminepalladium(II) hydrogen carbonate was tested for toxicity towards activated sludge of a predominantly domestic sewage. This GLP compliant study was conducted according to OECD test method 209.

In order to investigate the influence of the test substance towards activated sewage sludge, the inhibition of respiration was recorded after 30 minutes and 3 hours exposure time and compared to data for the control and a reference material, 3,5-dichlorophenol. Following a preliminary range-finding study, test concentrations of 10, 18, 32, 56 and 100 mg/l were selected for the definitive study.

The 3-hour EC50 for the test material, based on nominal test concentrations, was 35 mg/l. The validity criteria are fulfilled and the results are fully adequate for the assessment of this endpoint.

Reference 2

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.

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

no

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

The sludge was taken from the activation basin of the ESN (Stadtentsorgung Neustadt) sewage treatment plant D-67435 NW-Lachen-Speyerdorf and aerated during transport. Upon arrival at the test facility the sludge was filtered, washed with tap water and resuspended in tap water. The activated sludge was aerated until usage and fed daily with 50mL/L synthetic sewage feed.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Hardness:

1.08 mmol/L

Test temperature:

Pre-test: 19.1 - 20.8°C
Main study: 19.7 - 20.8°C

pH:

Pre-test: 6.25 - 8.25 at end of test
Main study: 7.6 - 8.0 at end of test

Dissolved oxygen:

Not reported

Salinity:

Not relevant

Nominal and measured concentrations:

Pre-test: Nominal Control, 1, 10, 100 and 100 mg/L
Main study: Nominal Control, 10, 20, 30, 60, 100 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Beaker
- Material, size, headspace, fill volume: Glass containing 500 mL test solution, nutrient solution and inoculum
- Aeration: purified air through pasteur pipette at 0.75L/min flow rate
- No. of vessels per concentration (replicates): Pre-test: 1 per test item concentration except highest contentration (5 replicates); Main study: 5 per test item concentration
- No. of vessels per control (replicates): Pre-test: 6 Control, 4 positive control; Main study: 6 Control, 4 positive control
- Biomass loading rate: Pre-test: 1.08g suspended solids/L; Main-study: 1.35 g suspended solids/L

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

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : oxygen consumption

TEST CONCENTRATIONS
- Spacing factor for test concentrations:
- Range finding study: yes
- Test concentrations: 0, 10, 100, 1000 mg/L
- Results used to determine the conditions for the definitive study: -4.2% inhibition at 1 mg/L, -0.6% inhibition at 10 mg/L, 81.7% inhibition at 100 mg/L, 100% inhibition at 1000 mg/L

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

29 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

Diamminedichloropalladium

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: 95% CL 28 - 30 mg/L

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

61 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

Diamminedichloropalladium

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: 95% CL 59 - 63 mg/L

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

18 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

Diamminedichloropalladium

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Results with reference substance (positive control):

3-hour EC50 8.8 mg/L (95% CL 5.0 - 13 mg/L) which lies within the recommended range of 2- 25 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 using and t-test or WEIR test.

Validity criteria fulfilled:

yes

Conclusions:

The 3-hour EC10 is 29 mg DDP/L (95% CL 28 - 30 mg/L). The 3-hour EC50 is 61 mg DDP/L (95% CL 59 - 63 mg/L). The NOEC is 18 mg DDP/L. This corresponds to 14.6 µg Pd L-1, 30.7 µg Pd L-1 and 9.1 µg Pd L-1, respectively.

Executive summary:

A GLP compliant, OECD guideline study to assess the inhibition of respiration of activated sludge was carried out (LAUS 2012). The study is considered to be suitable for use as the key study for this endpoint.

Reference 3

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 Sept 2018 (range finder) - 4 December 2018 (main test)

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

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

97.01% Tetraamminepalladium(2+) dichloride (Pd(NH3)4Cl2) based on analyzed Pd content of 42.06%, rest water

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

The test item was tetraamminepalladium dichloride (CAS 13815-17-3). The test item was added directly on a weight basis. The requisite mass to reach the desired concentrations was applied on pieces of Teflon, which were subsequently added to the dilution water. To achieve a maximum dilution of the test item, the assay was stirred overnight (following the indications for poorly water soluble substances in the "Leitfaden für Meldungen neuer Stoffe nach dem Chemikaliengesetz".

3,5-Dichlorophenol was used as reference item.

REPLICATES:
- Range finding experiment:
Inoculum blank: 6 vessels containing only inoculum (3 replicates were measured at the beginning and 3 at the end of the test assay series).
Test item: 1 vessel containing test item at 10 and 100 mg/L + inoculum.
Test item: 3 vessels containing test item at 1000 mg/L + inoculum.
Abiotic control: 1 vessel containing test item (1000 mg/L) only.
Reference item: 1 vessel containing reference item at 1, 6 and 36 mg/L + inoculum.

-main test:
Inoculum blank: 6 vessels containing only inoculum (3 replicates were measured at the beginning and 3 at the end of the test assay series).
Test item: 5 vessels containing test item at 3.60, 7.56, 18.87, 33.33 and 70.00 mg/L + inoculum.
Reference item: 1 vessel containing reference item at 1, 6 and 36 mg/L + inoculum.
(In the range finding test, only the effect of the test item on the total microbial respiration rate (total oxygen uptake including heterotrophic and nitrification activity) was investigated. Inhibition was found to be 28% at 10 mg/L and 95% at 100 mg/L. Therefore, a dose-response test with five concentrations was applied as main test.)

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

Fresh samples of activated sludge are withdrawn on September 17th, 2018 (range-finding test) and December 3rd, 2018 (main test) from the sewage treatment plant Ruhrverband Kläranlage, Sunthelle 6, 57392 Schmallenberg, Germany, which is mainly fed with municipal wastewater.

On return to the laboratory the sludge was left alone to settle and the supernatant was decanted. The sludge was washed one time with tap water. A small amount of the sludge was weighed and dried. From this a stock solution of activated sludge in tap water was prepared with a mixed liquor suspended solids level of about 3 – 4 g dry mass sludge/liter (range finding test 3.4 g/L, main test 3.6 g/L).

The sludge stock solution was fed with synthetic sewage feed and was incubated aerated overnight at 20 ± 2 °C in the dark. The amount of synthetic sewage feed applied was 25 mL/L. After this preincubation, the activated sludge was adjusted again on a mixed liquor suspended solids level of about 3 – 4 g dry mass sludge/liter (range finding test 3.3 g/L, main test 3.8 g/L). A respective volume of the adjusted sludge was applied into the test vessels to get a mixed liquor suspended solids level of nominally 1.5 g/L (range finding test 225 mL per 500 mL, main test 199 mL per 500 mL)

Test type:

static

Water media type:

freshwater

Remarks:

deionized water, free from inhibitory concentrations of toxic substances (e.g. Cu2+ ions) + synthetic sewage was used.

Limit test:

no

Total exposure duration:

3 h

Test temperature:

21-21.8 (range finding test)
19.8-21.5 (main test)

pH:

pH initial: 7.5 +/- 0.5

pH after 3h incubation:
8.4-8.6 (range finding test)
8.3-8.5 (main test)

Nominal and measured concentrations:

range finding test: 10, 100, 1000 mg/L
main test: 3.60, 7.56, 15.87, 33.33 and 70.00 mg/L

Test solutions not analysed, nominal concentrations only

Details on test conditions:

In each test vessel (1 L Erlenmeyer flasks) the respective amount of (i) test item, or (ii) reference item stock solution, dilution water, and synthetic sewage was applied. The total volume in the vessels was 500 mL, considering the volume of inoculum to be added to start the test. Before use, the pH was checked and buffered to the pH of 7.5 ± 0.5 using H2SO4 (1N) solution. The start pH was recorded.

In intervals of 5 - 15 minutes the inoculum was added . The content of the vessels was stirred and aerated (air flow 0.5 – 1 L/min) during incubation. The test was performed at 20 ± 2 °C. At the end of the test, the pH of each vessel was recorded again.

The respiration rates of the samples were measured 3 hours after start of incubation of the respective vessels. The respiration rates of an adequate aliquote of the samples (blank, test and reference assays) were measured and recorded over a period of 10 minutes or at least until an O2-content of 2.5 mg O2/L was reached. The measurements were carried out in glass beakers using an optical O2-sensor (FDO® 925 comined with an inoLab Multi 9630 measuring device by WTW Weilheim).

In the range finding and main tests, only the effect of the test item on the total microbial respiration rate (total oxygen uptake including heterotrophic and nitrification activity) was investigated.

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol (CAS-No. 591-35-5)

Key result

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

6 900 µg/L

Nominal / measured:

nominal

Conc. based on:

element

Basis for effect:

inhibition of total respiration

Remarks:

Inhibition based on total microbial respiration rate of the activated sludge

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

15.87 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

Inhibition based on total microbial respiration rate of the activated sludge

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

16.42 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

Inhibition based on total microbial respiration rate of the activated sludge

Remarks on result:

other: 95% Confidence Interval 11.18-20.73 mg/L

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

44.64 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

Inhibition based on total microbial respiration rate of the activated sludge

Remarks on result:

other: 95% Confidence Interval 39.04-51.60 mg/L

Duration:

3 h

Dose descriptor:

other: EC20

Effect conc.:

23.14 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

Inhibition based on total microbial respiration rate of the activated sludge

Remarks on result:

other: 95% Confidence Interval 17.66-27.56 mg/L

Details on results:

The total microbial respiration rate of the activated sludge in the control assays was found to be on average 22.29 mg O2/(L*h) after three hours. The total microbial respiration rate of the activated sludge incubated with concentrations of 3.60 to 70.00 mg TI per liter was on average between 5.49 and 20.37 mg O2/(L*h) after three hours.

Table 1: Mean respiration rate (mg O2/(h*g dry mass)).
TI = Test item; SD = Standard deviation; CV = Coefficient of variance.
Control 3.60 mg TI / L 7.56 mg TI / L 15.87 mg TI / L 33.33 mg TI / L 70.00 mg TI / L
Mean 22.29 20.37 20.85 19.30 15.70 5.49
SD 4.79 1.55 2.12 2.16 1.57 2.08
CV 21.5 7.6 10.2 11.2 10.0 38.0


Considering the data of all treatments from the main test, a dose-response relationship was observed. With 29.5% inhibition, a significant effect on the microbial respiration was observed at a concentration of 33.33 mg test item per liter. The NOEC was 15.87 mg TI/L.

Table2: Mean inhibition (%) of the respiration rate by the test item.
TI = Test item; *: significant difference when compared with control (p < 0.05).
3.60 mg TI / L 7.56 mg TI / L 15.87 mg TI / L 33.33 mg TI / L 70.00 mg TI / L
8.6 % 6.5 % 13.4 % 29.5 % * 75.4 % *


No significant abiotic oxygen demand by the test item was observed during the 3 h test period.

Results with reference substance (positive control):

The EC50 for the reference item was 17.71 mg/L and is therefore within the accepted range of the OECD 209 guideline of 2 to 25 mg/L;

Reported statistics and error estimates:

The respiration rates were calculated as mg O2/(g*h). The inhibitory effect of the test item on the respiration rate was expressed as a percentage of the mean respiration rates of six controls.

The oxygen uptake rates were calculated from the linear part of the graphs of oxygen concentration versus time, limiting the calculations to oxygen concentrations usually between 2.0 mg/L and 7.0 mg/L. However, since in some treatments respiration is heavily suppressed and consequently very slow and since the activated sludge applied respired very quickly, it was necessary to excurse into concentration bands below (or above) these values. This is - according to the guideline - acceptable since the extended sections of the uptake graph were straight and their gradients did not change as they pass through the 2.0 mg/L or 7.0 mg/L O2 boundaries.

The NOEC, LOEC, and, if possible, the EC10, EC20, EC50 and EC80 were determined. A LOEC was calculated by using ANOVA followed by Williams’ test. For trend analysis, a Contrasts test (Monotonicity of Concentration/Response) for metric data was applied. Since the test results show a concentration-response relationship, they were statistically analysed to determine EC values together with 95% confidence intervals using Probit-analysis assuming log-normal distribution of the values.

The evaluation of the concentration-effect-relationships and the calculations of effect concentrations were based on the nominal test item concentrations.
For statistical calculations, the computer program ToxRat Professional version 3.2.1 by ToxRat® Solutions GmbH was applied.

Validity criteria fulfilled:

yes

Remarks:

The test is considered valid, as the oxygen uptake rate in blank controls was ≥ 20 mg O2/g in an hour, the CoV of oxygen uptake rate in control replicates is ≤ 30% and the 3h-EC50 of the reference substance is in the accepted range of 2 to 25 mg/L

Conclusions:

A significant inhibition of bacterial respiration by Tetraamminepalladium(2+) dichloride (CAS 13815-17-3) was observed at a concentration of 33.33 mg per liter nominal concentration. The corresponding NOEC is 15.87 mg test item per liter. The EC10 and EC50 of Tetraamminepalladium dichloride (CAS 13815-17-3) are 16.42 mg test item per liter (95% CL: 11.18 – 20.73 mg/L) and 44.64 mg/L (95% CL: 39.04 – 51.60 mg/L), respectively, based on nominal concentrations. The corresponding EC10 is 6900 µg Pd/L.

Executive summary:

A study was performed to determine the effect of Tetraamminepalladium(2+) dichloride (CAS 13815-17-3) as test item (TI) on the total microbial respiration rate (total oxygen uptake including heterotrophic and nitrification activity) of an activated sludge according to the OECD guideline 209 and EC method C.11. As inoculum microorganisms from a sewage treatment plant mainly fed with municipal wastewater were used.

The test item was given to the synthetic sewage at a concentration of 3.60, 7.56, 18.87, 33.33 and 70.00 mg TI per liter. The vessels were stirred and aerated for 3 h at 20 ± 2 °C. Six replicates were prepared for controls (synthetic sewage and inoculum only), five replicates for the test item treated assays.

The total microbial respiration rate of the activated sludge incubated with concentrations of 3.60 to 70.00 mg TI per liter was found to be within 5.49 and 22.29 mg O₂/(L*h) after three hours. The respiration rate was significantly inhibited by the test item; starting at a concentration of 33.33 mg/L, TI significantly inhibited the respiration of the inoculum (respiration rate of 15.70 mg O₂/(L*h)). The NOEC was 15.87 mg TI per liter nominal concentration. The EC₁₀, EC₂₀and EC₅₀were determined at 16.42 mg TI per liter (CL 95: 11.18 – 20.73 mg/L), 23.14 mg TI per liter (CL 95: 17.66 – 27.56 mg/L) and 44.64 mg TI per liter (CL 95: 39.04 – 51.60 mg/L), respectively. The EC₈₀was higher than the highest concentration tested.

The corresponding EC10 is 6900 µg Pd/L.

Description of key information

The 3-hour EC50 for tetraamminepalladium hydrogen carbonate was determined to be 35 mg/L (12.56 mg Pd/L) (Mead and Worth, 1995).

The 3-hour EC50 for diamminedichloropalladium is 61 mg DDP/L (30.7 µg Pd/L). The NOEC is 18 mg DDP/L. ( 9.1 µg Pd/L) (Muckle, 2012).

The EC10 and EC50 of tetraamminepalladium dichloride are 16.42 mg test item/l and 44.64 mg/L, respectively. The corresponding EC10 is 6900 µg Pd/L (Simon, 2018).