calcium N2,N6-bis(3-carboxypropanoyl)lysine (1:1)

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:

From March 16th to 18th, 2016

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

Version / remarks:

adopted 22nd July 2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Remarks:

no analyses of the test concentrations were conducted.

Details on test solutions:

Since the test item is well soluble (325-350 g/l), the test concentrations were prepared by dilution, with natural water, of a stock solution prepared in natural water.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Source: non-adapted and not pre-conditioned activated sludge from the aeration tank of the ARA Werdhölzli (CH-8048 Zürich), a municipal biological waste water treatment plant.
- Sampling: 14th March 2016; 11.00 a.m.
- Preparation of inoculum for exposure: prior to the tests, the sludge was washed with natural water, and finally suspended in natural water at a concentration twice of the final concentration.
- Pretreatment: to keep the viability of the sludge, 80 ml of the synthetic sewage feed were added per litre of activated sludge (the volume taken into account is the volume of settled suspension) and the sludge was aerated overnight.
- Initial biomass concentration: 1.5 g/l dry matter in the final mixture.

Test type:

static

Water media type:

freshwater

Total exposure duration:

3 h

Test temperature:

22 ± 2 °C

pH:

All values were within an acceptable range.

Nominal and measured concentrations:

Nominal concentrations: 31.6, 100, 316 and 1000 mg/l.

Details on test conditions:

TEST SYSTEM
- Test vessel: 1000 ml glass flasks, containing a total volume of test suspension of 516 ml.
- Aeration: after addition of the activated sludge suspension, the test vessels were aerated by shaking (150 r.p.m.) for the whole test period of 3 hours.
- No. of vessels per concentration: activated sludge, test medium and test item, three replicates per test concentration.
- No. of vessels per blank control: activated sludge and test medium, six replicates.
- No. of vessels per sensitive control: activated sludge, test medium and 3,5-dichlorophenol as reference item (3, 10 and 30 mg/l), one vessel per test concentration.
- Sludge concentration: 3 g/l dry matter in the final mixture. 250 ml of the double-concentrated activated sludge suspension were added to each test vessel.

SYNTHETIC SEWAGE FEED
- Source/preparation: synthetic sewage feed prepared with ultra-pure water, as 100-fold sewage concentrate. 32 ml/l in the final mixture.
- Composition of synthetic sewage feed (dissolved amount per litre of ultra-pure water):
Peptone: 16 g
Meat extract: 11 g
Urea: 3 g
Sodium chloride: 0.7 g
Calcium chloride dihydrate: 0.4 g
Magnesium sulfate heptahydrate: 0.2 g
Dipotassium hydrogen phosphate: 2.8 g
- pH: the initial pH-value is 7.5 ± 0.5.
- Amount of synthetic sewage: 16 ml of 100-fold concentrated synthetic sewage were added to each test vessel.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: natural water supplying the laboratory.
- pH: 7.4 - 7.8
- Electric conductivity (room temperature): 477 - 536 μS/cm
- Total Hardness: 20.6 - 24.2 °f H; 2.06 - 2.42 mmol/l; 206 - 242 mg/l
- CO3 Hardness: 18.6 - 21.7 °f H; 186 - 217 mg/l
- Residual Hardness: 2.0 - 3.0 °f H; 20 - 30 mg/l
- Acid consumption: 3.72 - 4.34 mmol/l
- Colony counts (30°C): 0 - 1 per ml
- Escherichia coli: 0 per 100 ml
- Enterococcus: 0 per 100 ml
- Nitrate: 7.3 mg/l
- Chloride: 16.1 mg/l
- Sulfate: 18.6 mg/l

EFFECT PARAMETERS MEASURED
- Measuring apparatus: 300 ml flat bottom BOD flasks; polarographic oxygen electrode, connected to a potentiometric recorder.
- Sampling time: the respiration rate in each sample was measured 3 h after addition of the activated sludge suspension (following the starting sequence as described above), at 5–15 min intervals.
- Oxygen consumption: the respiration rate, R (in mg O2/l/h) is calculated from the linear part of the graphs of oxygen decrease versus time.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol (tested at 3, 10 and 30 mg/l)

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

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 total respiration

Details on results:

No significant inhibition of the activated sludge respiration, as compared to the untreated controls, was observed at any of the four concentrations tested.
Based on the data recorded, the median effect concentration EC50 of test item was estimated to be higher than 1000 mg/l nominal concentration.
The EC10, EC20, and EC80 values were also estimated to be higher than 1000 mg/l nominal concentration.
The no observed effect concentration (NOEC) was 1000 mg/l nominal concentration, as determined by Dunnett’s test.

VALIDITY OF THE TEST
All validity criteria were fulfilled.
- The oxygen uptake rate in the blank controls was 34.4 mg O2 per gram of activated sludge per hour (required >20 mg O2 per gram dry matter sludge per hour).
- The coefficient of variation of the oxygen uptake rate in the blank controls replicates was 5.7 % (required ≤ 30 %).
- The EC50 value of the reference item 3,5-dichlorophenol was estimated to be 6.4 mg/l (required 2–25 mg/l).

Results with reference substance (positive control):

The EC50 value of the reference item 3,5-dichlorophenol was estimated to be 6.4 mg/l (required 2 - 25 mg/l).

Activated sludge respiration rates after 3 h of exposure to the test and reference items and calculation of the inhibition

Nominal Concentration (mg/l)	Sample	O2 consumption rates (mg O2/l/h)	Respiration inhibition (%)	Respiration inhibition (%), Mean value
0	Blank	58.3	-	-
	Blank	51.5	-
	Blank	52.0	-
	Blank	49.5	-
	Blank	54.0	-
	Blank	52.5	-
3	3,5-dichlorophenol	37.4	29.4	29.4
10	3,5-dichlorophenol	19.0	64.1	64.1
30	3,5-dichlorophenol	6.4	87.9	87.9
31.6	Test item	48.9	7.6	2.6
	Test item	50.2	5.3
	Test item	55.7	-5.2
100	Test item	51.5	2.8	1.4
	Test item	52.4	1.2
	Test item	52.8	0.3
316	Test item	54.7	-3.3	1.3
	Test item	51.6	2.5
	Test item	50.5	4.7
1000	Test item	48.9	7.6	0.5
	Test item	53.7	-1.4
	Test item	55.4	-4.7

Validity criteria fulfilled:

yes

Conclusions:

EC50 (3h) > 1000 mg/l nominal concentration.
EC10, EC20, and EC80 (3h) > 1000 mg/l nominal concentration
NOEC (3h): 1000 mg/l nominal concentration, as determined by Dunnett’s test.

Executive summary:

Method

The inhibitory effect of test item on the oxygen consumption of activated sludge microorganisms of a municipal sewage treatment plant was investigated according to the OECD Guideline 209 under aerobic static conditions over an exposure time of 3 hours. The inhibitory effect may include the effect on respiration and nitrification.

The nominal concentrations tested were 31.6, 100, 316 and 1000 mg/l. Three parallel test vessels were used for each concentration of the test item and six vessels for the blank controls. No chemical analysis of test concentrations was conducted. For the calculation of the effective concentrations the nominal concentrations of the test item were used, assuming the test item to be stable in water over 3 h.

Results

No significant inhibition of the activated sludge respiration, as compared to the untreated controls, was observed at any of the four concentrations tested. Based on these data, the median effect concentration EC50 of test item was estimated to be > 1000 mg/l nominal concentration.

The EC10, EC20, and EC80, values of tested substance were also estimated to be > 1000 mg/l nominal concentration.

The no observed effect concentration (NOEC) was equal/higher than 1000 mg/l nominal concentration, as determined by Dunnett’s test.

Description of key information

EC50 (3h) > 1000 mg/l (nominal)

Key value for chemical safety assessment

EC50 for microorganisms:

1 000 mg/L

EC10 or NOEC for microorganisms:

1 000 mg/L

Additional information

The inhibitory effect of test item on the oxygen consumption of activated sludge microorganisms of a municipal sewage treatment plant was investigated according to the OECD Guideline 209 under aerobic static conditions over an exposure time of 3 hours. The inhibitory effect may include the effect on respiration and nitrification. The nominal concentrations tested were 31.6, 100, 316 and 1000 mg/l.

No significant inhibition of the activated sludge respiration, as compared to the untreated controls, was observed at any of the four concentrations tested. Based on the study outcomes, the median effect concentration EC50 of test item was estimated to be higher than 1000 mg/l nominal concentration.