Glycollic acid

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:

supporting study

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

GLP compliance:

yes

Analytical monitoring:

yes

Vehicle:

no

Details on test solutions:

1.0, 3.2, 10.32 and 100 mg/L glycolic acid 70%. An additional treatment containing 100 mg/L glycolic acid 70% but no inoculum, served as a physico-chemical control to check for abiotic oxygen uptake.

Test organisms (species):

activated sludge

Details on inoculum:

Secondary activated sludge from Wilmington, DE USA Publically Owned Treatment Works (POTW) was used as the microbial inoculum. The activated sludge was aerated and used within 24 h of collection.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Hardness:

Not applicable

Test temperature:

21.0 and 21.6˚C.

pH:

Not reported

Dissolved oxygen:

Test solutions were aerated with compressed air at a flow rate of approximately 0.5 to 1 liter per minute

Salinity:

Not applicable

Nominal and measured concentrations:

Nominal concentrations of 1.0, 3.2, 10, 32 and 100 mg/L glycolic acid 70%.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Duration:

3 h

Dose descriptor:

NOEC

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.:

> 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.:

> 70 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

70 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Details on results:

No further information

Results with reference substance (positive control):

The EC50 of the reference toxicant was 11 mg/L

Validity criteria fulfilled:

yes

Conclusions:

Following three hours exposure to glycolic acid 70% the EC50 was observed to be > 100 mg/L (equivalent to >70 mg a.s./L).

Executive summary:

The toxicity to bacteria of glycolic acid 70% was investigated according to guideline OECD 209 (1984). Following three hours exposure to glycolic acid 70% the EC50 was observed to be > 100 mg/L (equivalent to >70 mg glyolic acid/L) (based on nominal exposure concentrations).

Reference 2

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

5 January to 10 March, 2021

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 (Carbon and Ammonium Oxidation))

Version / remarks:

July 22, 2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Details on sampling:

Measurement of respiration rate:
A well-mixed sample of test medium from each test flask was poured into a Karlsruher flask after exactly 3 hours incubation and was not further aerated during measurement. Oxygen concentration was then measured with an oxygen electrode and recorded for approximately ten minutes, and the oxygen consumption (in mg O2 L-1 minute-1) subsequently determined. During measurement, the samples were continuously stirred on a magnetic stirrer.

Measurement of pH, dissolved oxygen and water temperature:
Oxygen concentrations and pH were determined at the start and at the end of the incubation period in at least one replicate of all test concentrations and controls. The water temperature was measured in one control medium at the start and the end of the incubation period.

Vehicle:

no

Details on test solutions:

Appropriate amounts of test item were directly weight into the test vessels, test water was added, and the resulting mixtures stirred intensively. Six controls (pure water, synthetic sewage feed and inoculum, but without addition of the test item) were tested in parallel. Stock Solution of N-allylthiourea (ATU): A nitrification inhibitor N-allylthiourea (ATU) was also tested on parallel in the same wa0,y with six separate controls and at the identical nominal concentrations of the test item and also the reference item 3,5-dichlorophenol under otherwise identical test conditions.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

Origin:
Activated sludge, microorganisms from a domestic wastewater treatment plant were supplied by a municipal sewage treatment plant (Bensheim, Germany).

Conditioning:
The activated sludge was used as collected, but coarse particles were removed by settling for a short period (15 minutes) and the upper layer then decanted. During holding prior to use, the sludge was fed with 50 mL synthetic sewage feed per litre and kept aerated at room temperature overnight. An aliquot of the final sludge suspension was weighed, dried and the ratio of wet sludge to its dry weight determined. Based on the sludge dry matter, calculated amounts of wet sludge were suspended in pure water to yield a concentration equivalent to 3 g/L on dry weight basis. This level gives a concentration of 1.5 g/L suspended solids in the test medium. The pH of the activated sludge inoculum was 6.8 and therefore no adjustment was necessary.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Remarks on exposure duration:

No further remarks

Post exposure observation period:

Not applicable

Hardness:

Not reported

Test temperature:

20 ± 2°C

pH:

3.1 to 8.2

Dissolved oxygen:

4.8 to 9.1 mg O2/L

Salinity:

Not applicable

Conductivity:

Not reported

Nominal and measured concentrations:

Nominal: 0 (control), 10, 32, 100, 320 and 1000 mg/L (without pH adjustment)

Details on test conditions:

Tests were conducted in glass flasks (ca. 1 litre volume) and Karlsruher flasks (300 mL volume) in a controlled environment room (20 ± 2°C) and aerated with compressed air (1.017 L/min). The room temperature was constantly recorded by a software-controlled temperature recording system (AMR Wincontrol).

For each replicate a test solution with a final volume of 500 mL was tested per treatment in a glass flask. Synthetic sewage feed (16 mL) and an adequate amount of the test item or an adequate volume of the stock solution of the reference item were initially filled up with pure water to 250 mL. At test initiation, activated sludge inoculum (250 mL) with a sludge concentration of 3.0 g/L suspended solids was added, first to the two controls, then to the reference item in increasing concentrations, then to two further controls, then to the test item in increasing concentrations and finally to the additional two controls. Four flasks were set up in parallel, two flasks for total respiration and two flasks of the corresponding treatment for heterotrophic respiration. After 15 minutes, the next four flasks were set-up. First two controls, then the test solutions of the reference item in increasing concentrations, then to two further controls, then the test item in increasing concentrations and finally to the additional two controls were set up for incubation for total and heterotrophic respiration. During the 3-hour aeration period the flasks were stirred on a magnetic stirrer to maintain sludge flocs in suspension.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol (tested at nominal test concentrations of 1, 4, and 16 mg/L, five replicates each, under otherwise identical test conditions)

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

320.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Duration:

3 h

Dose descriptor:

other: EC20

Effect conc.:

283.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

265.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

386.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of heterotrophic respiration

Duration:

3 h

Dose descriptor:

other: EC20

Effect conc.:

318 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of heterotrophic respiration

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

287.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of heterotrophic respiration

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of heterotrophic respiration

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

266.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of nitrification rate

Duration:

3 h

Dose descriptor:

other: EC20

Effect conc.:

241.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of nitrification rate

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

229 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of nitrification rate

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of nitrification rate

Duration:

3 h

Dose descriptor:

LOEC

Effect conc.:

320 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of nitrification rate

Details on results:

Effects on total respiration
Although the respiration rates in the 10 mg/L test item concentration flasks significantly diffed from the control, this had no biological relevance, as the respiration rates in the flasks treated with test item concentrations of 32 mg/L and 100 mg/L were not significantly different from the control. The respiration rates in the flasks treated with test item concentrations of 320 mg/L and 1000 mg/L showed significant differences compared to the control. Test item concentrations of 320 mg/L and 1000 mg/L showed inhibiting effects of about 50% and 100%, respectively, due to the increasing acidity of the test solution. The inhibitions were 11.5%, 3.2%, 0.3%, 51.3% and 99.3% for test item concentrations of 10, 32, 100, 320 and 1000 mg/L.

Effects on heterotrophic respiration:
The respiration rates in the flasks treated with test item concentrations between 10 mg/L and 1000 mg/L were significantly different from the control. In comparison to the inoculum controls the respiration was slightly inhibited by the two lowest test concentrations and increased with test concentrations up to a total inhibition at 1000 mg/L. The inhibition rates were 15.0%, 14.0%, 21.9%, 30.4% and 100.0% for test item concentrations of 10, 32, 100, 320 and 1000 mg/L, respectively. The heterotrophic respiration rates were statistically significantly different from the corresponding control rate in all the test item treatments, including the lowest tested concentration of 10 mg/L. However, the apparent effects on heterotrophic respiration at 10, 32 and 100 mg/L (below the threshold of the influence of adverse pH) were not dose-responsive and they were also not matched by similar effects on total respiration, where 67% of oxygen consumption was contributed by heterotrophic oxygen demand. The measurements of effect on heterotrophic respiration were made in treatments amended by addition of ATU and rely on the assumptions of the method that (1) ATU reliably suppressed only the nitrification component of total respiration and (2) no interaction occurred between ATU and the test item to cause additional impact on heterotrophic respiration above that caused by the test item alone. In view of the inconsistencies between effects observed in the tolerable pH range between total and heterotrophic respiration, the statistically significant effects on heterotrophic respiration are considered likely to be technical artefacts.

Effects on Nitrification:
The respiration rates in the flasks treated with test item concentrations of 10 mg/L, 32 mg/L and 100 mg/L were not significantly different from the control. The respiration rates in the flasks treated with test item concentrations of 320 mg/L and 1000 mg/L showed significant differences compared to the control. In comparison to the inoculum controls the total respiration rate of the activated sludge was not inhibited for test item concentrations up to and including 100 mg/L. Test item concentrations of 320 mg/L and 1000 mg/L showed inhibiting effects of about 100%. The inhibition was 4.4%, -18.3%,
-42.7%, 93.0% and 98.0% for test item concentrations of 10, 32, 100, 320 and 1000 mg/L, respectively.

Validity criteria:
The oxygen uptake in the untreated blank controls was 24.3 mg/g/h (i.e. > 20 mg/g/h) and the respiration rates of the controls did not differ by more than 30%. The observed values were 8.9% for the total respiration and 13.3% for the heterotrophic respiration. The validity criterion was therefore fulfilled.

Results with reference substance (positive control):

The inhibition of the activated sludge treated with the reference item was in the range of 16% to 45% for the test concentrations from 1 up to 16 mg/L. The 3-hour EC50 values of the reference item (3,5-dichlorophenol) for the total respiration, heterotrophic respiration without nitrification, and oxygen uptake due to nitrification were 21.2 mg/L, 18.0 mg/L and 0.2 mg/L, respectively. The EC50 values for the activated sludge batch used in the test were therefore within the appropriate validity ranges for total respiration, heterotrophic respiration without nitrification, and oxygen uptake due to nitrification (i.e. 2 to 25 mg/L, 5 to 40 mg/L and of 0.1 to 10 mg/L, respectively).

Reported statistics and error estimates:

ECx Estimation:
Effect concentrations (ECx values) for total respiration, heterotrophic respiration and nitrification were calculated via regression analysis using 3-Parametric normal CDF (cumulative distribution function), non-linear regression without weighting (optimization method: Levenberg-Marquardt). The 3-Parametric normal CDF R2 values for total respiration heterotrophic respiration and nitrification of the test item were 0.975, 0.916 and 0.842, respectively (thus a significant amount of variance is explained by the regression model in all three types of effect). The 3-Parametric normal CDF R2 values for total respiration heterotrophic respiration and nitrification of the reference substance (3,5-dichlorophenol) were 0.883, 0.758 and 0.353, respectively.

NOEC estimation:
For NOEC determination of the total respiration, heterotrophic respiration and nitrification effects, normal distribution was assessed via the Shapiro Wilk’s test (α= 0.01), variance homogeneity was assessed via the Levene’s Test (α= 0.01), and A T-test procedure for treatment comparison and NOEC estimation was performed via Williams Multiple Sequential Welsh t-test after Bonferroni-Holm (α= 0.05, one-sided smaller).

The software used to perform the statistical analysis was ToxRat Professional, Version 3.3.0® (ToxRat Solutions GmbH).

Influence of Glypure™ 99 and the reference item 3,5-Dichlorophenol on the inhibition of the total respiration rate of aerobic wastewater microorganisms after 3 hours of exposure (mean values)

Flask No.	Treatment	Conc. [mg/L]	Respiration rate [mean]^a [mg O2/g*h]	Standard deviation^a	Inhibition [mean]^a [%]	Significant effect compared to control^b
1, 2, 18, 19, 45, 46	Control	---	24.3	2.2	---	---
3-7	3,5-DCP	1	20.5	2.0	15.9	---
8-12	3,5-DCP	4	16.0	0.8	34.2	---
13-17	3,5-DCP	16	13.4	1.1	45.0	---
20-24	Test item	10	21.5	0.7	11.5	*
25-29	Test item	32	23.5	0.6	3.2	n.s.
30-34	Test item	100	24.2	1.3	0.3	n.s.
35-39	Test item	320	11.8	2.8	51.3	*
40-44	Test item	1000	0.2	0.2	99.3	*

Coefficient of variation of control: 8.9%

a: mean value of 5 replicates, 6 replicates in case of control

b: significance according to multiple sequentially-rejective Welch-t-test, one-sided smaller,α= 0.05 (* = significant; n.s.: not significant)

Inhibition [mean] % = 100 – (respiration rate [mean] [mg O₂/g*h] * 100 / mean value respiration rate in controls)

 

 

Influence of Glypure™ 99 and the reference item 3,5-Dichlorophenol on the inhibition of the heterotrophic respiration rate of aerobic wastewater microorganisms after 3 hours of exposure (mean values)

Flask No.	Treatment	Conc. [mg/L]	Respiration rate [mean]^a [mg O2/g*h]	Standard deviation^a	Inhibition [mean]^a [%]	Significant effect compared to control^e
47, 48, 64, 65, 91, 92	Control	---	16.2^d	2.2	---	---
49-53	3,5-DCP	1	13.6	0.6	16.0	---
54-58	3,5-DCP	4	13.9^c	0.6^c	14.3^c	---
59-63	3,5-DCP	16	7.9	1.0	51.1	---
66-70	Test item	10	13.8	1.1	15.0	*
71-75	Test item	32	13.9	0.3	14.0	*
76-80	Test item	100	12.6	0.7	21.9	*
81-85	Test item	320	11.3^b	1.5^b	30.4^b	*
86-90	Test item	1000	0.0	0.0	100.0	*

Coefficient of variation of control: 13.3%

a: mean value of 5 replicates, 6 replicates in case of control

b: mean value of 4 replicates due to an outlier

c: mean value of 3 replicates due to two outliers

d: this represents a reduction of 33.3% compared to the mean untreated control rate, indicating that nitrification-linked oxygen update suppressed by ATU addition contributed one third of the total respiration of the activated sludge used in this test

e: significance according to multiple sequentially-rejective Welch-t-test after Bonferroni-Holm, one sided smaller,α= 0.05 (* = significant; n.s.: not significant)

Inhibition [mean] % = 100 – (respiration rate [mean] [mg O₂/g*h] * 100 / mean value respiration rate in controls)

 

 

Influence of Glypure™ 99 and the reference item 3,5-Dichlorophenol on the inhibition of the nitrification respiration rate of aerobic wastewater microorganisms after 3 hours of exposure (mean values)

Treatment	Conc. [mg/L]	Respiration rate [mean]^a [mg O2/g*h]	Standard deviation^a	Inhibition [mean]^a [%]	Significant effect compared to control^b
Control	---	8.1	2.2	---	---
3,5-DCP	1	6.9	2.0	15.6	---
3,5-DCP	4	2.1	0.8	73.7	---
3,5-DCP	16	5.4	1.1	33.0	---
Test item	10	7.8	0.7	4.4	n.s.
Test item	32	9.6	0.6	-18.3	n.s.
Test item	100	11.6	1.3	-42.7	n.s.
Test item	320	0.6	2.8	93.0	*
Test item	1000	0.2	0.2	98.0	*

Coefficient of variation of control: 26.8%

a: mean value of 5 replicates, 6 replicates in case of control

b: significance according to multiple sequentially-rejective Welch-t-test after Bonferroni-Holm, one sided smaller,α= 0.05 (* = significant; n.s.: not significant)

Inhibition [mean] % = 100 – (respiration rate [mean] [mg O₂/g*h] * 100 / mean value respiration rate in controls)

Validity criteria fulfilled:

yes

Conclusions:

Glypure™ 99 (tested without pH adjustment) had no inhibiting effects on the respiration processes of a nitrifying activated sludge for test item concentrations up to and including 100 mg/L. Due to strong acidic properties of the test item, 320 mg/L and 1000 mg/L had strong inhibiting effects on total respiration, as well as its heterotrophic and nitrification components. The NOEC is therefore set at 100 mg/L. The 3-hour EC50 values were determined to be 320.6, 386.6 and 266.6 mg/L for inhibition of total respiration, heterotrophic respiration and nitrification rate, respectively.

Executive summary:

The toxicity of Glypure™ 99 to sewage treatment plant (STP) microorganisms in activated sludge, based on respiration inhibition, was assessed according to OECD Guideline 209.

 

Activated sludge from a predominately domestic sewage were exposed to test concentrations of 10, 32, 100, 320 and 1000 mg/L (without pH adjustment) and a control. All treatments were also prepared with additions of N-Allylthiourea (ATU, a specific inhibitor of nitrification) applied at a uniform concentration of 11.6 mg/L. The inhibition of total respiration, heterotrophic respiration and respiration based on nitrification in relation to control cultures was determined over a test period of 3-hours.

 

The oxygen uptake in the untreated blank controls was 24.3 mg/g/h (i.e. > 20 mg/g/h) and the respiration rates of the controls did not differ by more than 30%. The observed values were 8.9% for the total respiration and 13.3% for the heterotrophic respiration. The inhibition of the activated sludge treated with the reference item was in the range of 16% to 45% for the test concentrations from 1 up to 16 mg/L. The 3-hour EC50 values of the reference item (3,5-dichlorophenol) for the total respiration, heterotrophic respiration without nitrification, and oxygen uptake due to nitrification were 21.2 mg/L, 18.0 mg/L and 0.2 mg/L, respectively. The EC50 values for the activated sludge batch used in the test were therefore within the appropriate validity ranges for total respiration, heterotrophic respiration without nitrification, and oxygen uptake due to nitrification. The test was therefore considered valid.

 

Glypure™ 99 (without pH adjustment) had no inhibiting effects on the respiration processes of nitrifying activated sludge at test item concentrations ≥100 mg/L. Due to strong acidic properties of the test item, 320 mg/L and 1000 mg/L had strong inhibiting effects on total respiration, as well as its heterotrophic and nitrification components. The NOEC is therefore set at 100 mg/L. The 3-hour EC50 values were determined to be 320.6, 386.6 and 266.6 mg/L for inhibition of total respiration, heterotrophic respiration and nitrification rate, respectively.

Description of key information

Studies assessing the toxicity of glycolic acid on sewage treatment plant microorganisms are available. The key 3-hour EC50, EC10 and NOEC values (based on inhibition of nitrification respiration) were 266.6, 229.0 and 100 mg/L, respectively, shown in activated sludge microorganisms (Hammesfahr, 2021b). This key study was GLP compliant and met all validity criteria of the study guidelines followed (OECD Guideline 209, ISO 8192:2007 and EC 440/2008 Method C.11).

Key value for chemical safety assessment

EC50 for microorganisms:

266.6 mg/L

EC10 or NOEC for microorganisms:

100 mg/L