Hydrocotyle asiatica, ext.

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:

2019

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))

Version / remarks:

22 July 2010

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

Principles of method if other than guideline:

The following test item properties were investigated in the pre-experiment: the test item effect on the pH within the test system and the test item effect on the oxygen consumption of the activated sludge inoculum.
This preliminary test was designed according to the guideline requirements, triplicates were examined at the highest tested concentration of 1000 mg/L (as a limit concentration) and additionally two lower concentration levels of 10 and 100 mg/L were examined with one vessel each. Blank, abiotic (three abiotic controls with the highest concentration of the test item) and reference controls were included.
The nitrification potential of sludge was examined with additional control mixture (nitrification control in two parallels) that contained N-allylthiourea at 11.6 mg/L.
Because of more parallel running studies, the corresponding controls (blank, reference and nitrification) were commonly used.
The test media were prepared freshly before the test.

GLP compliance:

yes

Vehicle:

yes

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

Preparation of Activated Sludge Inoculum:
The coarse particles were removed by settling for 10 minutes, and the upper layer of finer solids was decanted. The activated sludge used for this study was washed by centrifugation and the supernatant liquid phase was decanted. The solid material was re-suspended in isotonic saline solution with shaking and again centrifuged. This procedure was repeated twice.
An aliquot of the final sludge suspension was weighed (5.394 g wet weight), dried and the ratio of wet sludge to dry weight (0.4222 g dry weight) determined. Based on this ratio, calculated amount of wet sludge (21 g dry weight that was equivalent to 268 g wet sludge) was suspended in isotonic saline solution (ad. 7 L) to yield a concentration equivalent to about 3 g per litre (on dry weight basis).
(In the test containers (300 mL final volume) the final concentration of suspended solids, containing 150 mL inoculum was 1.5 g per litre on dry weight basis.)
The above concentration calculation accounts for the dilution resulting from feeding with synthetic sewage. The activated sludge was not used on the day of the collection, but continuously aerated (2 L/minute) at the test temperature (20.0-20.8 oC) for about 48 hours (2 days) and fed daily with 50 mL synthetic sewage/L activated sludge.
The pH of the activated sludge inoculum was checked after preparation (pH: 7.38), additional pH adjustment of the inoculum was considered not necessary

Water media type:

freshwater

Total exposure duration:

3 h

Key result

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

ca. 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth inhibition

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

ca. 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth inhibition

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

ca. 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth inhibition

Details on results:

Validity of the Study
The specific respiration rate of the blank controls (without the test substance or reference substance) was 28.25 mg oxygen per one gram of activated sludge (dry weight of suspended solids) in an hour (higher than 20 mg/gh) with a coefficient of variation of 3.64 %.
The 3-hour EC50 of the reference item 3,5-Dichlorophenol (for the used activated sludge batch) was 14.60 mg/L within the range of 2 mg/L to 25 mg/L, that was required for total respiration (in this study the differentiation between heterotrophic respiration and nitrification was considered as not necessary).

Test Item Treatments

Defined amounts of the test item (1 x 3, 1 x 30, 3 x 300 mg in the test item treatments and 3 x 300 mg in the abiotic controls) were directly added in each test container, and after a pH measurement inoculated. The test item was investigated up to and including the concentration of 1000 mg/L without any pH adjustment before the inoculum addition. Analytical verification of test concentrations was not carried out.

In the experiment abiotic oxygen consumption of the test substance was not observed (in average 0.08 mg/Lh); therefore, the total oxygen consumption rates were not corrected with the abiotic control values in the subsequent calculations. The measured nitrification potential of the applied activated sludge (see nitrification control values) did not necessitate further testing. The influence of Centella asiatica dry extract on the oxygen consumption and specific respiration rate of activated sludge.
The observed oxygen consumption rates and consequently the specific respiration rates of the test item treatment groups were in the range of the blank controls. No significant inhibitory effect of the test item was observed. The observed, in average 3.67 % at 1000 mg/L inhibition was within the biological variability of the applied test system.
The specific respiration rates of the highest dose, 1000 mg/L were compared with the blank control values using 2 Sample t-Test (α=0.05). No statistically significant differences were observed in the comparison with the blank control values. Based on the results of this study the NOEC was determined to be 1000 mg/L.
Based on measured oxygen consumption values and calculated specific respiration rates it is concluded that the 3-hour EC10, EC50 and EC80 values of the test item are higher than 1000 mg/L.
The EC50 value was determined as: EC50 > 1000 mg/L.

Toxicity of 3,5-Dichlorophenol

The following concentrations of the positive reference control 3,5-Dichlorophenol were tested on the same activated sludge and under identical conditions as the test item: 2, 7 and 24.5 mg/L. In comparison to the blank controls the oxygen consumption rate of the activated sludge was inhibited by 11.34 % at the lowest concentration of 2 mg/L and at the nominal concentrations of 7 and 24.5 mg/L, the oxygen consumption rate was inhibited by 17.56 % and 71.12 %, respectively.
The 3-hour EC50 of 3,5-Dichlorophenol was calculated to be 14.60 mg/L.

Nitrification Controls

An additional nitrification control was examined in the test with two parallels to check the possible nitrification potential of the applied activated sludge batch. With the applying of the nitrification control the differentiation between the total, heterotrophic and nitrification respiration was possible. The total respiration (RT) was 42.38 mg/Lh, the heterotrophic respiration (RH) was 43.54 mg/Lh, the nitrification respiration (RN): 1.16 mg/Lh was calculated according to the equation: RN= RT-RH.
The obtained -1.16 mg/Lh was considered as not significantly different, being within a biological variability range of the applied test system, and lower than the 5 % of RT (2.12 mg/Lh) in blank controls.
According to the above calculations and based on the OECD 209 guideline it was assumed that the heterotrophic oxygen uptake equals the total uptake.

Environmental Conditions (pH, Temperature)

This test was performed without pH adjustment. The measured pH values in the prepared solutions: 3,5-Dichlorophenol reference control stock solution, synthetic sewage, activated sludge inoculum, prepared test mixtures (in the case of test item at the concentrations of 10 and 100 mg/L) etc. were in the acceptable pH 7-8 range at the start of the test. The measured pH values, the pH changes are summarized in Table.
In the test item containing test mixtures at the concentration of 1000 mg/L lower pH values were measured at the start of the test. The pH in the test containers containing 300 mg test item – water - synthetic sewage (1000 mg/L, see Table 2: T3/A T3/C) was pH ~ 6.5-6.8. After inoculation at these test item treatments the pH of T3/A and T3/B became within the required range of pH 7 – 8, the pH of T3/C remained slightly below the pH 7 - 8 range (pH: 6.86).
The test item may adversely affect pH within the test system; however, the results of the present preliminary test show, that the slightly lower pH caused by the test item has no effect on the applied inoculum culture and further testing (a definite test) with additional neutralization step of test item containing mixtures before inoculum addition is not necessary.

The mixtures were aerated at 0.5 L/min in the temperature range of 20±2o C. The temperature in the test mixtures during the measurements in average: 21.4o C, the measured minimum: 19.9o C, maximum: 22.0o C.

 

Table:The Oxygen Concentrations and pH Values in the Test Item Groups and Controls

Identifi-cation	Concentration (mg/L)	Oxygen concentration (mg O2/L)		pH
		Start of the 3-hour aeration	End of the 3-hour aeration	Before the addition of inoculum	Start of the 3-hour aeration	End of the 3-hour aeration
CBA	0.00	7.24	7.58	7.50	7.48	8.03
CBB		6.59	7.08	7.49	7.60	8.07
CBC		6.70	7.59	7.48	7.53	8.04
CBD		6.84	7.23	7.48	7.56	8.04
CBE		7.00	7.42	7.39	7.44	7.24
CBF		6.89	7.24	7.39	7.49	8.05
CBG		6.98	7.08	7.40	7.58	8.18
CBH		7.06	7.23	7.39	7.61	8.20
CNA	11.6 mg ATU/L	7.21	7.28	7.50	7.90	8.09
CNB		6.77	7.09	7.29	7.45	8.13
R1	2 mg 3,5-DCP/L	6.71	7.14	7.49	7.73	8.03
R2	7 mg 3,5-DCP/L	6.75	7.36	7.49	7.68	7.92
R3	24.5 mg 3,5-DCP/L	6.79	7.68	7.48	7.68	8.15
T1	10 mg Test Item/L	6.78	7.06	7.45	7.42	7.84
T2	100 mg Test Item/L	6.79	7.41	7.40	7.47	8.07
T3/A	1000 mg Test Item/L	6.59	7.07	6.81	7.35	7.82
T3/B		6.51	7.19	6.71	7.34	7.81
T3/C		6.88	7.11	6.50	6.86	7.77
CAA	1000 mg Test Item/L	6.74	8.38	‑ *	6.18	6.85
CAB		6.91	7.49	‑ *	6.26	6.77
CAC		6.70	8.12	‑ *	6.24	6.81

3,5-DCP:  3,5-dichlorophenol

ATU:          N-allylthiourea

Remarks:         *     At the Abiotic controls no inoculum was added; therefore one pH measurement was carried out at                    the start of the test.

Table:The Q1, Q2 and the applied Δt valuesin the Test Item and Control Groups; theOxygen Consumption Rate (R), and % Inhibition of R

Identifi-cation	Concentration (mg/L)	Oxygen concentration (mg O2/L)		Δt (min)	Oxygen Consumption Rate (R) (mg O2/Lh)	Average R (mg O2/Lh)	Inhibition of R (%)
		Q1	Q2
CBA	0.00	7.23	2.32	7	42.09	42.38	0.00
CBB		7.08	2.01	7.5	40.56
CBC		7.24	2.01	7.5	41.84
CBD		7.23	2.15	7.5	40.64
CBE		7.04	2.27	6.5	44.03
CBF		7.24	2.26	7	42.69
CBG		7.08	2.20	6.5	45.05
CBH		7.23	2.31	7	42.17
CNA	11.6 mg ATU/L	7.28	2.29	7	42.77	43.54	-2.73
CNB		7.09	2.29	6.5	44.31
R1	2 mg 3,5-DCP/L	7.14	2.13	8	37.58	37.58	11.34
R2	7 mg 3,5-DCP/L	7.07	2.12	8.5	34.94	34.94	17.56
R3	24.5 mg 3,5-DCP/L	7.68	5.64	10	12.24	12.24	71.12
T1	10 mg Test Item/L	7.06	2.01	7	43.29	43.29	-2.13
T2	100 mg Test Item/L	7.05	2.05	7	42.86	42.86	-1.12
T3/A	1000 mg Test Item/L	7.07	2.05	7.5	40.16	40.83	3.67
T3/B		7.19	2.29	7	42.00
T3/C		7.11	2.07	7.5	40.32
CAA	1000 mg Test Item/L	8.38	8.41	10	-0.18	0.08	99.81
CAB		7.49	7.48	10	0.06
CAC		8.12	8.06	10	0.36

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 these two measurements (min.).

3,5-DCP:  3,5-dichlorophenol

ATU:          N-allylthiourea

Table:The Specific Respiration Rate (R_S) in the Test Item and ControlGroups

Identification	Concentration (mg/L)	Specific Respiration Rate (mg O2/gh)	Average RS (mg O2/gh)
CBA	0.00	28.06	28.25 CV(%)= 3.64
CBB		27.04
CBC		27.89
CBD		27.09
CBE		29.35
CBF		28.46
CBG		30.03
CBH		28.11
CNA	11.6 mg ATU/L	28.51	29.03 CV(%)= 2.49
CNB		29.54
R1	2 mg 3,5-DCP/L	25.05	25.05
R2	7 mg 3,5-DCP/L	23.29	23.29
R3	24.5 mg 3,5-DCP/L	8.16	8.16
T1	10 mg Test Item/L	28.86	28.86
T2	100 mg Test Item/L	28.57	28.57
T3/A	1000 mg Test Item/L	26.77	27.22^NS CV(%)= 2.50
T3/B		28.00
T3/C		26.88
CAA	1000 mg Test Item/L	-0.12	0.05 CV(%)= 338.19^#
CAB		0.04
CAC		0.24

3,5-DCP:  3,5-dichlorophenol

ATU:          N-allylthiourea

CV:            Coefficient of variation

^NS:    There is no statisticallysignificant difference when compared to the control (2-Sample t-Test; 2-sided; α = 0.05)

^#:      The high CV value was caused by the low calculated mean value and a standard deviation of 0.18.

 

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the performed Activated Sludge Respiration Inhibition Test, the EC10 and EC50 values of test item was determined as higher than 1000 mg/L. Based on the statistical evaluation in this test the NOEC was 1000 mg/L. This pre-test demonstrated the absence of inhibition of oxygen consumption of the test item up to and including the limit concentration of 1000 mg/L, therefore in line with OECD 209 guideline an additional definite test is not required.

Description of key information

The EC10 and EC50 values of test item was determined as higher than 1000 mg/L. Based on the statistical evaluation in this test the NOEC was 1000 mg/L.

Key value for chemical safety assessment

EC50 for microorganisms:

1 000 mg/L

EC10 or NOEC for microorganisms:

1 000 mg/L