Glycollic acid

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

4 June to 6 August, 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Version / remarks:

October 2, 2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Duplicate samples from the freshly prepared test media of all test concentrations and the control were taken at the test start (day 0) and at a test medium renewal period in the second and third week (days 10 and 17). For the determination of the stability of the test item under the test conditions and of the maintenance of the test item concentrations during the test media renewal periods, duplicate samples of the respective aged test media and controls were sampled at days 3, 12 and 19. One of these three stability samplings was over a test medium renewal period of 72 hours (weekend), two of a renewal period of 48 hours. All samples remained undiluted until sample preparation. All samples were stored in a freezer (≤ -20°C), protected from light until analysis was performed. Afterwards the samples were again stored deep frozen and will be kept stored up to the date of the final report.

Vehicle:

no

Details on test solutions:

The test water was Elendt "M4" reconstituted water, prepared by dissolving the appropriate nomical amounts of analytical grade salts and additives in deionised water.

Before test start and before every test medium renewal, the test medium of the highest test concentration of nominal 100 mg/L was prepared by dissolving; 147.1 mg test item into 1471 mL test water (day 0), 157.1 mg test item into 1571 mL test water (day 3), 155.8 mg test item into 1558 mL test water (day 5), 158.6 mg test item into 1586 mL test water (day 7), 156.1 mg test item into 1561 mL test water (day 10), 161.4 mg test item into 1614 mL test water (day 12), 160.8 mg test item into 1608 mL test water (day 14), 158.1 mg test item into 1581 mL test water (day 17), 158.8 mg test item into 1588 mL test water (day 19), respectively, by intense stirring for 15 to 25 minutes. The pH of the test medium at test start and every test water renewal was adjusted
from pH 4.0 - 4.1 to 7.9 - 8.5 using 2 M NaOH. Adequate volumes of these test media were diluted with test water to prepare the test media of the desired test concentrations. The test media were prepared just before introduction of Daphnia (= start of the test) and each test medium renewal. The appearance of the test item in the test media was determined in all freshly prepared and aged test media of all test concentrations. There were no remarkable observations.

Test organisms (species):

Daphnia magna

Details on test organisms:

Source: in-house laboratory culture;
Age at test start: 1.75 to 22.50 hours old;
Sex: female;
Breeding conditions: The Daphnia were bred in the test laboratory under similar temperature and light conditions as in the test. The cultivation of the parental Daphnia was performed in Elendt M4 medium. The test organisms were not first brood progeny. The Daphnia in the stock culture were fed at least all working days with green algae (Desmodesmus subspicatus) freshly grown in the test laboratory;
Acclimation: Not necessary, since the test was performed in the same medium as the culturing

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Remarks on exposure duration:

No further remarks

Post exposure observation period:

Not applicable

Hardness:

195.8 to 213.6 mg CaCO3/L

Test temperature:

19.9 to 20.7 °C in the freshly prepared media;
20.1 to 20.8 °C in the aged media

pH:

7.8 to 8.3 in the freshly prepared media;
7.6 to 8.8 in the aged media

Dissolved oxygen:

8.6 to 8.9 mg O2/L in the freshly prepared media;
7.7 to 10.0 mg O2/L in the aged media

Salinity:

Not applicable

Conductivity:

Not reported

Nominal and measured concentrations:

Nominal concentrations: 0 (control), 1.0, 3.2, 10, 32, 100 mg/L;
Time-weighted average measured concentrations: 0.668, 2.02, 5.69, 24.5, 89.6 mg/L

Details on test conditions:

Tests were conducted in 100 ml volume glass beakers containing ca. 60 mL test medium. Tests were performed with 10 replicates per treatment group, with 1 Daphnia introduced per replicate.

The test media of all test concentrations and of the control were renewed on Days 3, 5, 7, 10, 12, 14, 17 and 19 of the exposure period (every Monday, Wednesday and Friday). At these dates the test Daphnia were carefully transferred from test vessels with aged test media into new test vessels with freshly prepared test media of the corresponding concentrations. The handling and the allocation of the treatments was randomised on every test media renewal.

The test Daphnia were fed daily with green algae of the species Desmodesmus subspicatus, freshly grown in the test laboratory. The amount of food was based on the concentration of total organic carbon (TOC) in the algal food suspension.

Tests were conducted within a controlled environment room. The pH-values, dissolved oxygen concentrations and water temperatures were measured in the freshly prepared test media of all treatment groups with suitable instruments at test start and then once a week. The corresponding aged test media of all treatment groups were measured. The water hardness was determined once a week in the control and the highest tested concentration in fresh and aged test media. A photoperiod of 16:8 hours light:darkness (light intensity: 320 to 670 Lux).

Observations on mortality of adults and offspring occurrence were made daily. The number of offspring was counted at each water renewal and determined for each day. Dead offspring were counted if possible at each water renewal. Dead adults and offspring were removed at each water renewal day. The day of first eggs, of first brood and the number of offspring per adult in each treatment replicate was documented. Additionally, any signs of intoxication (e.g. pale, small adults, aborted eggs) were recorded.

Reference substance (positive control):

not specified

Key result

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

> 89.6 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: Reproduction per introduced adult / Reproduction per surviving adult / Adult mortality

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

>= 89.6 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: Reproduction per introduced adult / Reproduction per surviving adult / Adult mortality

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

> 89.6 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: Reproduction per introduced adult / Reproduction per surviving adult / Adult mortality

Duration:

21 d

Dose descriptor:

other: EC20

Effect conc.:

> 89.6 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: Reproduction per introduced adult / Reproduction per surviving adult / Adult mortality

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

> 89.6 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: Reproduction per introduced adult / Reproduction per surviving adult / Adult mortality

Details on results:

Mortality of adults:
The Fisher’s exact binominal test revealed no significant trend in mortality. At concentrations of 2.02, 5.69 and 89.6 mg test item/L, all Daphnia survived until the end of the test. At test concentrations of 0.668 and 24.5 mg test item/L, 1 animal died during the test. In the control, 2 Daphnia died during the course of the test

Reproduction:
the total number of live offspring produced per introduced and surviving adult within 21 days of continuous exposure to Glypure™ 99 (cumulative values) are presented in the results tables presented under ‘Any other information on results incl. tables’.

Observations:
In addition to the reported adult mortality and the production of live juveniles, dead offspring were also observed during the test. The numbers of dead offspring were well distributed over all concentrations and no concentration dependency could be observed. The dead offspring and all other observation made on Daphnia such as small or pale appearance, aborted eggs and the appearance of first eggs in the brood pouch are given in Tables 5 to 10. According to the Guideline, dead offspring are not included in the cumulative counts of juvenile production, where only live offspring are considered.

Analytical results:
In the freshly prepared test media, 101% of the nominal test concentrations were found (average of all test concentrations). In the aged test media, 48% of nominal were found (average of all test concentrations). All reported results refer to time weighted average concentrations, since the test item concentrations were not within ± 20% of the nominal concentrations during the test.

Validity criteria:
The validity criteria of the study were met, as adult Daphnia mortality in the control did not exceed 20% at the end of the test (20% mortality was observed) and the mean number of live offspring in the control after 21 days was > 60 per surviving adult Daphnia (80 per surviving adult Daphnia). The coefficient of variation around the mean number of living offspring produced per surviving adult Daphnia in the control was 27%.

Results with reference substance (positive control):

Not applicable

Reported statistics and error estimates:

The mortality of adult daphnia was tested with the Cochran-Armitage test on a concentration-response pattern. Replicate 10 of the control, replicate 8 of concentration 0.668 mg test item/L and replicate 6 of concentration 24.5 mg test item/L were excluded from all analysis, because the Daphnia died (isolated instances of non-substance related mortality with no known cause). Since a Cochran-Armitage trend test revealed no significant trend in mortality, the evaluation can be based on reproduction (total number of live offspring) per surviving adult. The data for reproduction (total number of live offspring) per introduced adult are also evaluated and reported. The NOEC and the LOEC for reproduction per introduced and surviving adult were determined by Dunnett´s t-test. The NOEC and the LOEC for mortality were determined by Fisher’s exact binominal test. The EC50, EC20 and EC10 with their 95 %-confidence limits for mortality could not be calculated due to absence of toxicity of the test item. Therefore, ECx values were determined directly from the raw data. The software used to perform the statistical analysis was ToxRat Professional, Version 3.3.0, ToxRat Solutions GmbH.

Number of surviving adult Daphnia magna exposed to test item

Exposure day	Time-weighted average concentration [mg test item/L]
	Control	0.668	2.02	5.69	24.5	89.6
0	10	10	10	10	10	10
1	10	10	10	10	10	10
2	10	10	10	10	10	10
3	10	10	10	10	10	10
4	10	10	10	10	9	10
5	10	10	10	10	9	10
6	10	10	10	10	9	10
7	10	10	10	10	9	10
8	10	10	10	10	9	10
9	10	10	10	10	9	10
10	9	10	10	10	9	10
11	9	10	10	10	9	10
12	9	10	10	10	9	10
13	9	10	10	10	9	10
14	9	10	10	10	9	10
15	9	10	10	10	9	10
16	9	10	10	10	9	10
17	9	10	10	10	9	10
18	8	10	10	10	9	10
19	8	9	10	10	9	10
20	8	9	10	10	9	10
21	8	9	10	10	9	10
No. introduced	10	10	10	10	10	10
Dead Daphnia	2	1	0	0	1	0
% mortality	20	10	0	0	10	0

No significant difference from the control(tested with Fisher`s Exact Binomial Test with Bonferroni- Correction,a = 0.05, one-sided greater)

 

  

Cumulative number of live offspring produced by all introduced adults per treatment within 21 days of exposure

Exposure day	Time-weighted average concentration [mg test item/L]
	Control	0.668	2.02	5.69	24.5	89.6
0	0	0	0	0	0	0
1	0	0	0	0	0	0
2	0	0	0	0	0	0
3	0	0	0	0	0	0
4	0	0	0	0	0	0
5	0	0	0	0	0	0
6	0	0	0	0	0	0
7	0	9	0	0	0	0
8	0	9	0	0	0	0
9	2	10	4	21	15	3
10	15	95	65	93	39	60
11	41	120	88	110	70	69
12	55	128	88	159	117	110
13	139	267	216	266	164	208
14	165	333	252	332	277	251
15	197	333	252	361	282	256
16	266	508	415	462	341	375
17	357	603	462	557	476	429
18	404	603	462	571	476	459
19	484	758	632	695	575	607
20	592	840	738	811	720	674
21	658	840	738	850	740	718
No. introduced	10	10	10	10	10	10
Mean No. of offspring per introduced adult	65.8	84.0	73.8	85.0	74.0	71.8

In this table the live offspring of all introduced adultDaphniais displayed including the live offspring ofDaphniawhich died during the course of the test.

  

 

Cumulative number of live offspring produced by all surviving adults per treatment within 21 days of exposure

Exposure day	Time-weighted average concentration [mg test item/L]
	Control	0.668	2.02	5.69	24.5	89.6
0	0	0	0	0	0	0
1	0	0	0	0	0	0
2	0	0	0	0	0	0
3	0	0	0	0	0	0
4	0	0	0	0	0	0
5	0	0	0	0	0	0
6	0	0	0	0	0	0
7	0	9	0	0	0	0
8	0	9	0	0	0	0
9	2	10	4	21	15	3
10	15	95	65	93	39	60
11	36	114	88	110	70	69
12	50	122	88	159	117	110
13	134	261	216	266	164	208
14	160	313	252	332	277	251
15	192	313	252	361	282	256
16	261	488	415	462	341	375
17	338	561	462	557	476	429
18	385	561	462	571	476	459
19	465	716	632	695	575	607
20	573	798	738	811	720	674
21	639	798	738	850	740	718
No. introduced	8	9	10	10	9	10
Mean No. of offspring per introduced adult	79.9	88.7	73.8	85.0	82.2	71.8

In this table the number of live offspring of all adultDaphniawhich survived until the end of the test is displayed.

  

  

Number of live offspring produced by each individual adult after 21 days of exposure

Replicate	Time-weighted average concentration [mg test item/L]
	Control	0.668	2.02	5.69	24.5	89.6
1	91	79	78	123	109	57
2	66	80	63	83	69	71
3	19	159	91	120	81	75
4	54	53	85	60	84	105
5	100	97	75	96	95	79
6	114	85	65	51	0	67
7	86	116	89	71	50	73
8	73	42	60	65	91	55
9	55	63	66	91	97	83
10	0	66	66	90	64	53
Mean	65.8	84.0	73.8	85.0	74.0	71.8
SD	35.5	33.9	11.4	24.1	31.3	15.5
CV	54.0%	40.4%	15.5%	28.4%	42.3%	21.6%
% inhibition	-	-27.7	-12.2	-29.2	-12.5	-9.1

SD: standard deviation, CV: coefficient of variance
No significant difference from the control(tested with Dunnett’st-test,a = 0.05, one-sided smaller)

  

 

Number of live offspring produced by each surviving adult after 21 days of exposure

Replicate	Time-weighted average concentration [mg test item/L]
	Control	0.668	2.02	5.69	24.5	89.6
1	91	79	78	123	109	57
2	66	80	63	83	69	71
3	-	159	91	120	81	75
4	54	53	85	60	84	105
5	100	97	75	96	95	79
6	114	85	65	51	-	67
7	86	116	89	71	50	73
8	73	-	60	65	91	55
9	55	63	66	91	97	83
10	-	66	66	90	64	53
Mean	79.9	88.7	73.8	85.0	82.2	71.8
SD	21.6	32.4	11.4	24.1	18.5	15.5
CV	27.0%	36.5%	15.5%	28.4%	22.4%	21.6%
% inhibition	-	-11.0	7.6	-6.4	-2.9	10.1

SD: standard deviation, CV: coefficient of variance, -: adult died during the course of the test
No significant difference from the control(tested with Dunnett’s t-testt,a = 0.05, one-sided smaller)

  

 

Summary of analytical results

Sample Description	fresh (0h)			aged (48/72h)			Time Weighted Average^2
	% of	RSD		% of	RSD
[mg test item/L]	nominal^1	[%]	n	nominal^1	[%]	n	[%]	[mg test item/L]^3
Control	n.a.	n.a.	6	n.a.	n.a.	6	n.a.	n.a.
1.0	108	3	6	38	1	6	67	0.668
3.2	103	3	6	35	103	6	63	2.02
10	100	2	6	32	137	6	57	5.69
32	98	1	6	55	53	6	77	24.5
100	96	3	6	82	12	6	90	89.6

¹mean value of all measured samples per treatment group

²Time weighted average calculated according to OECD Guidance Document No. 23, Annex 2

³Values were rounded to three significant digits
RSD: relative standard deviation per treatment group
n: number of analysed samples
n.a.: not applicable

Validity criteria fulfilled:

yes

Conclusions:

The 21 day NOEC was determined to be ≥ 89.6 mg test item/L for reproduction per introduced adult and ≥ 89.6 mg test item/L for reproduction per surviving adult. The 21 day NOEC was determined to be ≥ 89.6 mg test item/L for mortality of the adult test animals.
The EC10 was determined to be > 89.6 mg test item/L for reproduction per introduced adult and > 89.6 mg test item/L for reproduction per surviving adult after 21 days. The EC10 was determined to be > 89.6 mg test item/L for mortality.
The EC50 for reproduction per introduced adult was determined to be > 89.6 mg test item/L and > 89.6 mg test item/L for reproduction per surviving adult. The EC50 for mortality was determined to be > 89.6 mg test item/L.

Executive summary:

The effects of Glypure™ 99 on survival and reproduction per introduced and per surviving adult of Daphnia magna were determined in a semi-static test with an exposure period of 21 days, according to OECD guideline 211. The pH of the test medium at test start and every test water renewal was adjusted from pH 4.0 - 4.1 to 7.9 - 8.5 using 2 M NaOH.

 

All reported results refer to time weighted average concentrations, since the test item concentrations were not within ± 20% of the nominal concentrations during the test. The validity criteria of the study were met, as adult Daphnia mortality in the control did not exceed 20% at the end of the test (20% mortality was observed) and the mean number of live offspring in the control after 21 days was > 60 per surviving adult Daphnia (80 per surviving adult Daphnia).

 

The 21‑day NOEC was determined to be ≥89.6 mg test item/L for reproduction per introduced adult and ≥89.6 mg test item/L for reproduction per surviving adult. The 21‑day NOEC was determined to be≥89.6mg test item/L for mortality of the adult test animals. The EC₁₀ was determined to be > 89.6 mg test item/L for reproduction per introduced adult and > 89.6 mg test item/L for reproduction per surviving adult after 21 days. The EC₁₀ was determined to be> 89.6mg test item/L for mortality. The EC₅₀ for reproduction per introduced adult was determined to be> 89.6mg test item/L and > 89.6 mg test item/L for reproduction per surviving adult. The EC₅₀ for mortality was determined to be> 89.6mg test item/L.

Description of key information

One experimental study assessing the chronic toxicity of glycolic acid to aquatic invertebrates is available. The effects of Glypure™ 99 on survival and reproduction per introduced and per surviving adult of Daphnia magna were determined in a semi-static test with an exposure period of 21 days. No effects were observed in this study up to the highest nominal test concentration of 100 mg/L, corresponding to the time weighted average concentration of 89.6 mg/L. Therefore, the key 21d-NOEC value based on measured concentrations was ≥89.6 mg/L for reproduction per introduced adult, reproduction per surviving adult and mortality of the adult test animals. In the same way, the key 21d-EC10 value based on measured concentrations was >89.6 mg/L for reproduction per introduced adult, reproduction per surviving adult and mortality of the adult test animals. This key study was GLP compliant and met all validity criteria of the study guideline (OECD guideline 211).