Propylenediamine

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:

18-NOV-2020 to 09-DEC-2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

The concentration of the test substance was determined in one representative test interval per
week in each freshly prepared test solution (test vessels without daphnids) and in the 48-h or
72-h old test solutions before renewal (combined sample from test vessels with daphnids).
Moreover, samples were analyzed from the 48-hour or 72-hour old abiotic replicate (no
Daphnia or food algae) per test group.
For each concentration, two retained samples were taken at each time point. Retained
samples are stored at the Laboratory ECO in a freezer (at approx. -20°C). Analysis of these
samples were performed in case of equivocal analytical results with the original samples or
after loss of/damage to original samples after agreement by the Study Director.

Vehicle:

no

Details on test solutions:

The stock test solution (12 mg/L) was prepared by dissolving 60 mg of test substance in 5 L of test medium and stirring for approximately 10 min at room temperature. The lower test concentrations were prepared by dilution of this stock solution. Before the dilution was performed, the stock solution was checked for complete dissolution of the test substance.
Before use, pH of the stock solutions was adjusted to control media pH (8.0) with 1 mol HCl.

In a preliminary test without a GLP-status, the test substance was stable in the test medium over a period of 96 hours. The test solutions were visibly homogeneous.


Test solution renewal: Semi-static, renewal 3 times per week

Test organisms (species):

Daphnia magna

Details on test organisms:

Test species: Daphnia magna STRAUS

Reason for selection ofthe test species:
Recommended species in the test guidelines

Origin:
The clone of Daphnia magna STRAUS 1820 used was supplied by the Institut National de Recherche Chimique Appliquée, France, in 1978. From this date on this clone was cultured and bred continuously in the Ecotoxicology Laboratory of Experimental Toxicology and Ecology, BASF SE, Ludwigshafen Germany.

Culture conditions:
Daphnia brood stock are kept in mass cultures consisting of approx. 20–30 individuals for a maximum of 4 weeks. All individuals in the mass culture originate from a single female. After approximately 14 days the adults have produced at least 3 broods and the young can be used in tests. Offspring are
removed from the mass cultures at least once daily during the normal work week to ensure that young daphnia are <24-h old (first instar) at start of exposure. Detailed records are kept (in test facility archives) to monitor the health of Daphnia brood stock cultures including observations of young production, mortality, ephippia, and measurement of water chemistry parameters. Only young from healthy cultures without signs of stress are used for testing.

Acclimatization:
The Daphnia are cultured under the identical conditions as the
test including test media (Elendt M4), water quality, temperature
(20 ±1°C), and diet.

Age at start of exposure:
< 24 h

Test type:

semi-static

Water media type:

freshwater

Remarks:

A synthetic fresh water (Elendt M4) is used as media for culture and test purposes. For the composition of this M4 medium see OECD 211.

Limit test:

no

Total exposure duration:

21 d

Hardness:

See Table 1

Test temperature:

see Table 2

pH:

see Table 3

Before use, pH of the stock solutions was adjusted to control media pH (8.0) with 1 mol HCl.

Dissolved oxygen:

see Table 4

Conductivity:

550-650 µS/cm

Nominal and measured concentrations:

Method of analysis: HPLC-MS
Limit of quantification (LOQ): 0.1 mg/L
The nominal concentrations were 0 mg/L, 0.7 mg/L, 1.5 mg/L, 3 mg/L, 6 mg/L and 12 mg/L

For detailed results of the measured concentrations in the test biotic containers see Table 5, for the abiotic samples see Table 6.

All measured concentrations deviated less than 20% from the nominal concentration. Thus, the test substance was stable in solution under test conditions and nominal concentrations are an accurate representation of exposure levels maintained throughout the test period. Following recommendations in OECD 211 the results should be evaluated based on the nominal concentrations.

Additionally, samples were analyzed from the abiotic replicate of each test concentration and the control at the end of the renewal interval. All abiotic replicates were within ±20% of the nominal concentrations. These samples were analyzed to assist in the interpretation of results in the event that food algae or Daphnia over the exposure period interfered with the test substance analyses.

Details on test conditions:

TEST SYSTEM
- Test vessel: Numbered polypropylene cups (nominal volume 100 mL), filled with approx. 120 mL test solution (i.e. reduced headspace), and closed with screw caps (due to the potential volatility of the test item).
- Aeration: no
- Renewal rate of test solution: 3 times per week
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10

- Biomass loading rate:
During the test daphnids were fed daily a diet of live green algae Desmodesmus subspicatus, cultured in a synthetic medium. The algae were separated from their culture medium by centrifugation, resuspended in daphnid's medium (M4)
corresponding to concentrations of 1790 mg and 2060 mg TOC*/L (respectively) in the algal concentrates used. The daphnids were fed a defined volume (≤ 112 μL) of the concentrate. The algae were stored in a refrigerator (dark, about 4-8°C) for maximum 21 days. By adding the algal concentrate, the test solution was slightly diluted. During a worst case 72-h test interval with 3 feedings,
0.112 mL each, a total of 0.336 mL are added to ~120 mL test volume resulting in a maximal dilution of ~0.3%.

*amount of feed algae determined as total organic carbon

OTHER TEST CONDITIONS
- Photoperiod: 16h light 8h darkness
- Light intensity: 710 - 740 lux, wavelngth 400 - 750 nm

Reference substance (positive control):

yes

Remarks:

The test with sodium chloride is carried out every month at the testing facility.

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

> 12 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: All meas. conc. deviated less than 20% from nominal concentration. Thus, the test substance was stable in solution under test conditions and nominal concentrations are an accurate representation of exposure levels maintained throughout the test period.

Details on results:

No significant mortality or any other additional significant adverse effects or abnormal behavior
were observed in any of the test treatments.

Results with reference substance (positive control):

- Results with reference substance valid? yes
The EC 50 (48 h) was 4.81 g/L. Thus, the daphnia are responding normally to toxic stress.

Reported statistics and error estimates:

The following parameters and statistical procedures were used to evaluate significant differences to the control and determine the LOEC and NOEC:
- Reproduction as number of living young (Wilcoxon’s test, one sided with Bonferroni Holm adjustment, one sided analysis)
- *Parent mortality as immobilized organisms (Fishers exact test, one sided analysis)
- % immobile young (Wilcoxon’s test, with Bonferroni Holm adjustment, one sided analysis)
- % aborted eggs (Wilcoxon’s test, one sided with Bonferroni Holm adjustment, one sided
analysis)
- Time to first brood (Wilcoxon’s test, with Bonferroni Holm adjustment, one sided analysis)
- Growth as length in mm (Dunnett´s test, one sided analysis)

The NOEC and LOEC are based on the primary population relevant parameters identified in the OECD test guideline [Ref. 2], reproduction and mortality.
Reproduction is reported based on surviving and introduced daphnids according to the recommendations in the OECD guideline [Ref. 2]. The percent reduction of number of living young, the percent reduction of length and percent increase in time to first brood were calculated in relation to the control mean. Percentages lower than 0 were set to 0. The percent immobile young in each test group was calculated in relation to the sum of the number of immobile and number of living young. The percent aborted eggs in each test group was
calculated in relation to the sum of number of aborted eggs, number of immobile and number of living young. The endpoints length, time to first brood, immobile young and aborted eggs were evaluated only for organisms that survived to the end of the test.

*Was not evaluated, as no parent mortality was observed in any of the test groups throughout the study.

Table 5: Nominal and measured concentrations of the test item in the respective treatments.

 Nominal Concentration [mg/L]

	Interval days	Analytically Measured Concentration (biotic)
		Initial		Old ^a		Overall Mean		Mean initial
		[mg/L]	[%]b	[mg/L]	[%]b	[mg/L]	[%]b	[mg/L]	[%]c
0 (Control)	Day 0 - 2	n.a.	-	n.a.	-	-	-	-	-
	Day 9 - 12	n.a.	-	n.a.	-
	Day 19 - 21	n.a.	-	n.a.	-
0.7	Day 0 - 2	0.61	95%	0.61	95%	0.64	100%	0.64	91%
	Day 9 - 12	0.65	102%	0.67	105%
	Day 19 - 21	0.66	104%	0.65	101%
1.5	Day 0 - 2	1.36	98%	1.38	100%	1.40	101%	1.38	92%
	Day 9 - 12	1.40	101%	1.46	106%
	Day 19 - 21	1.39	101%	1.43	103%
3	Day 0 - 2	2.77	96%	2.77	95%	2.85	98%	2.90	97%
	Day 9 - 12	2.85	98%	2.96	102%
	Day 19 - 21	3.09	106%	2.67	92%
6	Day 0 - 2	5.38	91%	5.83	99%	6.02	102%	5.91	99%
	Day 9 - 12	5.71	96%	6.14	104%
	Day 19 - 21	6.65	112%	6.39	108%
12	Day 0 - 2	11.10	92%	11.40	95%	12.10	101%	12.03	100%
	Day 9 - 12	11.70	97%	12.20	101%
	Day 19 - 21	13.27	110%	12.93	108%

a: 24 hours old

b: % of mean Initial

c: % of nominal concentration

n.a. not assigned, no significant peak detected

 

Table 6: Nominal and measured concentrations of the abiotic samples of the respective treatment.

Nominal Concentration [mg/L]	Interval days	Analytically Measured Concentration (abiotic)
		Initial		Old ^a		Overall Mean		Mean initial
		[mg/L]	[%]b	[mg/L]	[%]b	[mg/L]	[%]b	[mg/L]	[%]c
0 (Control)	Day 0 - 2	n.a.	-	n.a.	-	-	-	-	-
	Day 9 - 12	n.a.	-	n.a.	-
	Day 19 - 21	n.a.	-	n.a.	-
0.7	Day 0 - 2	0.61	95%	0.60	93%	0.64	100%	0.64	91%
	Day 9 - 12	0.65	102%	0.66	104%
	Day 19 - 21	0.66	104%	0.66	103%
1.5	Day 0 - 2	1.36	98%	1.44	104%	1.41	102%	1.38	92%
	Day 9 - 12	1.40	101%	1.48	107%
	Day 19 - 21	1.39	101%	1.40	101%
3	Day 0 - 2	2.77	96%	2.70	93%	2.88	99%	2.90	97%
	Day 9 - 12	2.85	98%	3.06	105%
	Day 19 - 21	3.09	106%	2.80	96%
6	Day 0 - 2	5.38	91%	5.40	91%	5.92	100%	5.91	99%
	Day 9 - 12	5.71	96%	6.25	106%
	Day 19 - 21	6.65	112%	6.16	104%
12	Day 0 - 2	11.10	92%	11.43	95%	11.89	99%	12.03	100%
	Day 9 - 12	11.70	97%	11.75	98%
	Day 19 - 21	13.27	110%	12.09	101%

a: 24 hours old

b: % of mean Initial

c: % of nominal concentration

n.a. not assigned, no significant peak detected

 

Table 7: Mortality and Reproduction Summary after 21 days

Test groups	Nominal concentration [mg/L]	Mortality	Reproduction per surviving parent		Reproduction per introduced parent
		Parent animals	Mean Living Young	% effectb	Mean Living Young	% effectb
0	0 (control)	0	135.4 (14.5%a)	–	135.4 (14.5%a)	–
1	0.7	0	139.3	–	139.3	–
2	1.5	0	133.0	–	133.0	–
3	3	0	136.5	–	136.5	–
4	6	0	128.5	–	128.5	–
5	12	0	132.6	–	132.6	–
–: Not measured or evaluated. a: Coefficient of variation for control fecundity based on surviving parents. b: Effect relative to control. Only calculated for statistically significant effects.

 

Table 8: Other biological observations among surviving parent animals after 21 days

Nominal concentration [mg/L]	Mean growth (length, mm)	% Immobile young	Mean days to first brood	% Aborted eggs
0 (control)	4.35	0	9.5	0.2
0.7	4.38	0.1	9.4	0.5
1.5	4.40	0	9.5	1**
3	4.25	0	9.5	0.5
6	4.29	0.1	9.6	0.7
12	4.35	0.1	9	1*

Statistical significance: *= p≤ 0.05, **= p≤ 0.01

Validity criteria fulfilled:

yes

Conclusions:

No significant mortality or any other additional significant adverse effects or abnormal behavior were observed in any of the test treatments.

The test substance did not have a chronic toxic effect on Daphnia magna up to 12 mg/L.

Since the analytically determined concentrations of the test substance in the test solutions were within ±20% of the nominal concentrations, the effect concentration can be expressed relative to the nominal concentration for the evaluation of the test substance.

The results in this study are consistent with all validity criteria and the test is valid according to the guidelines of this study. No deviations from test guidelines or other incidents occurred during the course of the reported test which may have influenced the results.

Description of key information

In a 21-day chronic toxicity study, Daphnia magna were exposed to 1,2-Propylendiamine to nominal concentrations of 0 (control) and 0.7, 1.5, 3, 6, and 12 mg/L under semi-static conditions in accordance with the OECD 211 guideline. The reprodutcion of the daphnids was not effected by the test item. Therefore, the NOEC is >12 mg/L. Additionally, no significant mortality or any other significant adverse effects or abnormal behavior were observed in any of the test treatments.

 

 

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

NOEC

Effect concentration:

> 12 mg/L

Additional information

In a 21-day chronic toxicity study,Daphnia magnawere exposed to1,2-Propylendiamineto nominal concentrations of 0 (control) and 0.7, 1.5, 3, 6, and 12 mg/L under semi-static conditions in accordance with the OECD 211 guideline. The reprodutcion of the daphnids was not effected by the test item. Therefore, the NOEC is >12 mg/L. Additionally, no significant mortality or any other significant adverse effects or abnormal behavior were observed in any of the test treatments.

 

 

It can be concluded that long-term effects on aquatic invertebrates are not expected.