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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 Sept 2018 (range finder) - 11 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:
>99.9% Palladium (II) di(4-oxopent-2-en-2-oate; Pd(C5H7O2)2) based on analysed Pd content of 34.94%
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
The test item was Palladium (II) di(4-oxopent-2-en-2-oate) (CAS 14024-61-4). 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 referecne 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 5.14, 10.80, 22.68, 47.62 and 100.00 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 8% at 10 mg/L and 69% 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 10th, 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 3.4 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 3.3 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 225 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)
19.1-20.8 (main test)
pH:
pH initial: 7.5 +/- 0.5

pH after 3h incubation:
8.3-8.4 (range finding test)
8.1-8.4 (main test)
Nominal and measured concentrations:
range finding test: 10, 100, 1000 mg/L
main test: 5.14, 10.80, 22.68, 47.62, 100 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)
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
5.14 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
Key result
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
6.68 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 3.33-10.26 mg/L
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
119.25 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 Value extrapolated by the statistical software (maximum nominal dose level = 100 mg/L)
Remarks on result:
other: 95% Confidence Interval 87.6-191.03* mg/L (*: value extrapolated by the statistical software)
Duration:
3 h
Dose descriptor:
other: EC20
Effect conc.:
17.97 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 12.15-23.55 mg/L
Details on results:
The total microbial respiration rate of the activated sludge in the control assays was found to be on average 24.46 mg O2/(L*h) after three hours. The total microbial respiration rate of the activated sludge incubated with concentrations of 5.14 to 100.00 mg TI per liter was on average between 14.06 and 24.82 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 5.14 mg TI / L 10.80 mg TI / L 22.68 mg TI / L 47.62 mg TI / L 100.00 mg TI / L
Mean 24.46 24.82 21.74 17.35 15.22 14.06
SD 2.26 2.67 1.80 0.72 2.25 0.57
CV 9.2 10.7 8.3 4.2 14.8 4.0

Considering the data of all treatments from the main test, a dose-response relationship was observed. With 11.1% inhibition, a significant effect on the microbial respiration was observed at a concentration of 10.80 mg test item per liter. The NOEC was 5.14 mg/L.

Table2: Mean inhibition (%) of the respiration rate by the test item.
TI = Test item; *: significant difference when compared with control (p < 0.05).
5.14 mg TI / L 10.80 mg TI / L 22.68 mg TI / L 47.62 mg TI / L 100.00 mg TI / L
8.6 % 6.5 % 13.4 % 29.5 % * 7 5.4 % *

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 24.9 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 Palladium (II) di(4-oxopent-2-en-2-oate) (CAS 14024-61-4) was observed at a concentration of 10.80 mg per liter nominal concentration. The NOEC is 5.14 mg test item per liter. The EC10 and EC50 of Palladium (II) di(4-oxopent-2-en-2-oate) (CAS 14024-61-4) are 6.68 mg test item per liter (95% CL: 3.33 – 10.26 mg/L) and 119.25 mg/L (95% CL: 87.60 – 191.03 mg/L), respectively, based on nominal concentration.
Executive summary:

A study was performed to determine the effect of Palladium (II) di(4-oxopent-2-en-2-oate) (CAS 14024-61-4) 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 5.14, 10.80, 22.68, 47.62 and 100.00 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 5.14 to 100.00 mg TI per liter was found to be within 14.06 and 24.46 mg O2/(L*h) after three hours. The respiration rate was significantly inhibited by the test item; starting at a concentration of 10.80 mg/L, TI significantly inhibited the respiration of the inoculum (respiration rate of 21.74 mg O2/(L*h)). The NOEC was found to be 5.14 mg TI per liter nominal concentration. The EC10, EC20and EC50were determined at 6.68 mg TI per liter (CL 95: 3.33 – 10.26 mg/L), 17.97 mg TI per liter (CL 95: 12.15 – 23.55 mg/L) and 119.25 mg TI per liter (CL 95: 87.60 – 191.03 mg/L), respectively. The EC50value and its upper 95%-CL value are extrapolated by the statistic software. The EC80was found to be higher than the highest concentration tested.

Description of key information

The EC10 and EC50 were determined at 6.68 and 119.25 mg TI per liter.

Key value for chemical safety assessment

EC50 for microorganisms:
119 250 µg/L
EC10 or NOEC for microorganisms:
6 680 µg/L

Additional information

A study was performed to determine the effect of Palladium (II) di(4-oxopent-2-en-2-oate) (CAS 14024-61-4) 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 5.14, 10.80, 22.68, 47.62 and 100.00 mg TI per liter. The EC10 and EC50 were determined at 6.68 mg TI per liter (CL 95: 3.33 – 10.26 mg/L) and 119.25 mg TI per liter (CL 95: 87.60 – 191.03 mg/L), respectively.