Fatty acids, tall-oil, compds. with (Z)-N-9-octadecenyl-1,3-propanediamine (2:1)

Long-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

31st August 2018 to 12th November 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Version / remarks:

2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Additiv 309

IUPAC Name:

Additiv 309

Test material form:

liquid: viscous

Details on test material:

Identification: Optimol Additiv 309
Appearance: Pasty dark brown liquid
Batch: 0000771660
Purity/Composition: UVCB
Test item storage: At room temperature
Stable under storage conditions until: 27 June 2021 (expiry date)

Specific details on test material used for the study:

Test item information

Identification: Optimol Additiv 309
Appearance: Pasty dark brown liquid
Batch: 0000771660
Purity/Composition: UVCB
Test item storage: At room temperature
Stable under storage conditions until: 27 June 2021 (expiry date)

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Sampling for Analysis of Test Concentrations:

Samples for analysis were taken from all test concentrations and the control.

Sampling: Frequency - At the beginning and at the end of three intervals of 48 hours (nominal days 5 and 7, 12 and 14, 19 and 21) and one interval of 72 hours (nominal days 0 and 3).
Volume - 2.0 mL
Storage - Samples were stored in a freezer (≤-15°C) until analysis at the analytical laboratory of the Test Facility.

At the end of the refreshment period, the replicates were pooled at each concentration before sampling.

Additionally, reserve samples of 2.0 mL were taken from all test solutions for possible analysis. If not already used, these samples were stored in a freezer (≤-15°C) for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis.

Test solutions

Vehicle:

no

Details on test solutions:

The batch of Optimol Additiv 309 tested was a pasty dark brown liquid UVCB substance, not completely soluble in test medium at the loading rates initially prepared. No correction was made for the purity/composition of the test item.

Preparation of test solutions started with loading rates individually prepared in the range of 1.0 to 100 mg/L. A 3-day period of magnetic stirring was applied to accelerate dissolution of the test item in medium. The obtained mixtures were allowed to settle for approximately one day period . Thereafter, the aqueous Water Accommodated Fractions (WAFs) were collected by means of siphoning and used as test concentrations. The control received the same treatment. All test solutions were clear and colorless at the end of the preparation procedure.

Any residual volumes were discarded. The preparation procedure was performed at room temperatures.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST SYSTEM
Species: Daphnia magna (Crustacea, Cladocera) (Straus,1820), at least third generation, obtained by acyclical parthenogenesis under specified breeding conditions.

Source: In-house laboratory culture with a known history.

Reason for selection: This system has been selected as an internationally accepted invertebrate species.

Validity of batch: Daphnids should originate from a healthy stock, 2nd to 5th brood, showing no signs of stress such as mortality >20%, presence of males, ephippia or discoloured animals and there should be no
delay in the production of the first brood.

Characteristics: To initiate the test, young daphnids < 24 hours old are selected, from parental daphnids, preferably individually housed, and greater than two weeks old.

BREEDING
Start of each batch: With new-born daphnids, i.e. less than 3 days old, by placing them individually in 50 mL M7medium or in a bulk culture by placing about 250 daphnids into 5 litres of medium in an all-glass culture vessel.

Maximum age of the cultures: 4 weeks

Renewal of the bulk cultures: After 7 days of cultivation half of the medium twice a week.

Temperature of medium: 18-22°C

Feeding: Daily, a suspension of fresh water algae (Chlorella sp.).

Validity of the cultures: Historical data on the reproductive capacity are based on the numbers of living young in the individual cultures and tested to meet the validity criteria for survival and reproduction.

Medium: M7, as prescribed by Dr. Elendt-Schneider (Elendt, B.-P., 1990: Selenium deficiency in Crustacea. An ultrastructural approach to antennal damage in Daphnia magna Straus.Protoplasma 154, 25-33).

Composition of medium M7:
Adjusted ISO medium: the following chemicals (analytical grade) are dissolved in tap water purified by Reverse Osmosis (RO-water, GEON Waterbehandeling, Berkel-Enschot, The Netherlands):

Macro salts: CaCl2.2H2O 211.5 mg/L
MgSO4.7H2O 88.8 mg/L
NaHCO3 46.7 mg/L
KCl 4.2 mg/L

The following salts and vitamins were added to freshly prepared ISO medium to reach the following concentrations:

Salts: H3BO3 0.71mg/L
FeSO4.7H2O 0.25mg/L
MnCl2.4H2O 0.090mg/L
LiCl 0.076mg/L
RbCl 0.018mg/L
SrCl2.6H2O 0.038mg/L
Na2MoO4.2H2O 0.015mg/L
NaBr 0.0040mg/L
CuCl2.2H2O 0.0042mg/L
ZnCl2 0.013mg/L
CoCl2.6H2O 0.010mg/L
KI 0.0032mg/L
Na2SeO3 0.0022mg/L
NH4VO3 0.00057mg/L
Na2EDTA.2H2O 0.62mg/L
Na2SiO3.5H2O 7.5mg/L
NaNO3 0.27mg/L
KH2PO4 0.14mg/L
K2HPO4 0.18mg/L
Vitamins: Thiamine hydrochloride 75.0µg/L
B12 1.0µg/L
Biotin 0.75µg/L

The hardness: 180 mg/L expressed as CaCO3 and the pH between 6 and 9.

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Test conditions

Hardness:

>140mg/L (as CaCO3)

Test temperature:

18-22 degrees C, constant within 2 degrees C

pH:

Between 6.0 and 9.0, constant within 1.5 units

Dissolved oxygen:

>3mg/L

Details on test conditions:

TEST PROCEDURE AND CONDITIONS

Test duration : 21 days

Test type: Semi-static

Frequency of renewal: Three times a week

Test vessels Volume: 60 ml (6 x Ø 3.5 cm), all-glass covered with a Perspex plate.

Medium: M7

Experimental design: At the start of the experiment (nominal day 0) 10 neonate daphnids, less than one day old, per group were divided over ten vessels each containing a minimum of 50 ml test medium. The control group consisted of 20 daphnids.

Light: 16 h photoperiod daily. Light intensity should not exceed 1000-1500 lux which corresponds with a range not exceeding 15-20 µE.m-2.s-1.

Temperature: 18-22°C, constant within 2°C

Oxygen concentration: > 3 mg/L

pH: Between 6.0 to 9.0, constant within 1.5 units.

Feeding: In general twice daily an amount of 0.25 ml of a Chlorella sp. suspension. On weekend days and on day 0 and 4 of exposure, an amount of 0.50 ml was added in one single feed. This daily ration corresponded to 0.2 mg C/Daphnia/day, which is the recommended value for daily feeding per daphnid in the reproduction test according to the OECD Guideline 211.

TESTING STRATEGY AND EXPERIMENTAL DESIGN

Range Finding Test:
A preliminary test was performed prior to the reproduction test. WAFs prepared at 1.0, 10 and 100 mg Optimol Additiv 309 per litre were tested. A control group was also included. Each concentration consisted of two replicates containing a total of ten daphnids (five each). The total test period was ten days. Test conditions were held as similar as possible to those applied in the reproduction test. Feeding rate was slightly reduced in order not to obscure observations.

Test solutions were renewed on days 1, 3 and 6 during the test. Samples for possible analysis were taken at days 0 (fresh), 1 (fresh and old), 3 (fresh and old) and 6 (old). Samples were taken from solutions containing the daphnia and food (algae), but also from an extra vessel incubated under the same conditions, but without daphnia and algae.

Reproduction Test:
Optimol Additiv 309: Individual WAFs prepared at loading rates of 1.0, 1.8, 3.2, 5.6 and 10 mg/L

Controls: Test medium without test item or other additives

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

21 d

Dose descriptor:

NOELR

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Effect concentrations 2

Key result

Duration:

21 d

Dose descriptor:

NOELR

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Effect concentrations 3

Key result

Duration:

21 d

Dose descriptor:

NOELR

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth

Details on results:

RANGE-FINDING TEST

Table 1 summarizes the daily observations of mortality and reproductive potency of surviving parental daphnids during the 10-day range-finding test.

No mortality of parental daphnids was observed in the control and WAFs prepared at 1.0 and 10 mg/L during the exposure period. In these groups, the first appearance of eggs in the brood pouch was recorded at the same time. None of the daphnids exposed to the highest loading rate survived day 5 of the exposure.

m/z 325.3: the measured concentrations were clearly higher at the loading rate of 10 mg/L than at 1.0 mg/L. In all refreshment periods a decrease of measured concentrations was observed. The observed decline of concentrations was similar in samples incubated with daphnia and algae as in their abiotic counterparts. This indicates that the observed decrease was not caused by biological processes.

m/z 589.7: In most of the cases, the concentrations measured in WAF prepared ta loading rate of 10 mg/L gave higher responses than at the loading rate of 1.0 mg/L. However, no clear trend in stability was observed (17-213% of initial values were measured at the end of the refreshment period).

It should be noted that the concentration of both components measured in freshly prepared WAF of 10 mg/L at day 3 of exposure, was two orders of magnitude higher than on other occasions. The reason for this is unknown.
Based on above and the knowledge that the test item is a UVCB substance, it was decided to perform the reproduction test with refreshment of test solutions three times a week.

Test conditions during the range-finding test were maintained within the limits prescribed by the study plan.

REPRODUCTION TEST

Measured concentrations:
Samples taken during this study were analysed for two components. Table 2 shows the calculated time weighted average (TWA) concentrations of both components.

m/z 325.3: Measured concentrations differed at different sampling occasions, however, a dose-response trend was always observed. The calculated TWA concentrations also increase with increasing loading rate. This indicates that the WAFs were prepared correctly.

m/z 589.7: The measured concentration showed high degree of scattering and no clear dose-dependency. Also the calculated TWA concentrations did not showed dose-dependency. This component is believed to be very poorly soluble, while m/z 325.3 is a surface active substance. Therefore, it is possible, that some of the micelles formed by m/z 325.3 contained particles of m/z589.7 causing this scattering of results

Because the test item is a UVCB substance with low solubility, the effect parameters were express in terms of loading rates initially prepared.

Condition of Parental Daphnids:
Two out of the twenty parental daphnids died during the test period in the control treatment (10%; see Table 3). Hence, parental mortality did not exceed 20% in the control. Three daphnids did not survive exposure in the lowest WAF, and one daphnid did not survive exposure in the highest WAF. No mortality was observed in the WAFs prepared at 1.8, 3.2 and 5.6 mg/L. Hence, the mortality of the parental daphnids was clearly not treatment related. This was confirmed by the trend analysis. Therefore, the effects on reproduction were calculated based on number of offspring produced by surviving parental daphnids.

Time to First Reproduction:
The first offspring were observed on day 7 in all treatments.

Reproduction:
Group mean cumulative numbers of offspring per surviving parental daphnids at the end of the test are shown in Table 4.

On average, 142 offspring were produced per surviving daphnid in the control treatment. In all groups the number of offspring was higher than 117, which is double the requirement for the control treatment. In two groups statistically significant reduction of reproduction was observed (15 and 18% in WAFs prepared at 1.8 and 5.6 mg/L, respectively), however, the observed effects were not dose-related nor attributable to any of measured components. For this reason, and the fact that at the highest loading rate, i.e. the regulatory limit loading rate, no significant effect was observed, it was considered justified to set the NOEL for reproduction on 10 mg/L.

A number of immobilised offspring was observed during the test. The observations were not dose-related and therefore considered not relevant (see Table 5). No observations of offspring trapped at the surface of the solutions or aborted eggs were made in this study.

Body Length:
The group mean body length of the surviving daphnids per concentration measured at the end of the test and the relative reduction of body length compared to the control are summarised in Table 6.
The effects on growth ranged from 2.0% reduction to 1.2% of increase and were not dose-related. Statistical analysis showed that none of these effects were significant.

Determination of Effect Concentrations:
Table 7 shows the effect parameters based on loading rates.

EXPERIMENTAL CONDITIONS

Exposure parameters are summarized in Table 8.

The pH was maintained within the limits prescribed by the study plan (6.0-9.0, constant within 1.5 units).

The oxygen concentration complied with the requirements as laid down in the study plan (> 3 mg/L).

The temperatures complied with the requirements as laid down in the study plan (18-22°C, constant within 2°C).

Total hardness complied with the requirements as laid down in the study plan (>140 mg CaCO3 per liter).

Light intensity complied with the recommendations of the guideline, i.e. was below 1000-1500 lux.

The total dissolved organic carbon content of the M7 medium complied with the value recommended by the guidelines (i.e. was < 2 mg/L).

Any other information on results incl. tables

Table1     
Survival and Reproductive Potency During the Range-Finding Test

Day	Optimol Additiv 309, Loading rate (mg /L)
	Control				1.0				10				100
	A		B		A		B		A		B		A		B
	P	F1	P	F1	P	F1	P	F1	P	F1	P	F1	P	F1	P	F1
0	5	-	5	-	5	-	5	-	5	-	5	-	5	-	5	-
1	5	-	5	-	5	-	5	-	5	-	5	-	2	-	3	-
2	5	-	5	-	5	-	5	-	5	-	5	-	0	n.a.	1	n.a.
3	5	-	5	-	5	-	5	-	5	-	5	-	0	n.a.	1	n.a.
4	5	-	5	-	5	-	5	-	5	-	5	-	0	n.a.	1	n.a.
5	5	(5)	5	(5)	5	(5)	5	(5)	5	(5)	5	(5)	0	n.a.	1	n.a.
6	5	(5)	5	(5)	5	(5)	5	(5)	5	(5)	5	(5)	0	n.a.	0	n.a.
7	5	29	5	(5)	5	(5)	5	(5)	5	37	5	(5)	0	n.a.	0	n.a.
8	5	25	5	51	5	44	5	41	5	23	5	43	0	n.a.	0	n.a.
9	5	(5)	5	(5)	5	(5)	5	(5)	5	(5)	5	(5)	0	n.a.	0	n.a.
10	5	5	5	(5)	5	(5)	5	(5)	5	(5)	5	(5)	0	n.a.	0	n.a.
Group total	110				85				103				0

 ( )number of parents with eggs in their brood pouch, n.a. – not applicable as no surviving parental daphnids

Table2          
Nominal and Time Weighted Mean Exposure Concentrations

Optimol Additiv 309 Loading rate (mg/L)	Time Weighted Mean Exposure Concentration (mg/L)
	m/z 325.3	m/z 589.7
1.0	0.0052	0.00027
1.8	0.0069	0.00306
3.2	0.008	0.00040
5.6	0.045	0.00372
10	0.12	0.00351

 

Table3          
Mortality (Immobility) of Parental Daphnids at the End of the Test

Optimol Additiv 309 Loading rate (mg/L)		Introduced		Mobile		Immobile		%Immobility
Control		20		18		2		10
1.0		10		7		3		30
1.8		10		10		0		0
3.2		10		10		0		0
5.6		10		10		0		0
10		10		9		1		10

Table4          
Group Mean Cumulative Number of Juveniles per Surviving Parent and Reduction of Reproduction at the End of the Test

Optimol Additiv 309 Loading rate (mg/L)	Mean	Std. Dev.	n	%Reduction
Control	142.3	19.14	18
1.0	140.4	21.51	7	1.3
1.8	121.3	24.95	10	15*
3.2	133.3	15.04	10	6.3
5.6	116.8	22.87	10	18*
10	137.4	17.26	9	3.4

* Statistically significant (p<0.05)

Table5          
Incidence of Immobilised Offspring

Optimol Additiv 309 Loading rate (mg/L)	Immobile offspring
Control	0
1.0	3
1.8	0
3.2	2
5.6	8
10	0

Table6          
Group Mean Body Lengths (mm) and Reduction of Growth of Parental Daphnids at the End of the Test

Optimol Additiv 309 Loading rate (mg/L)	Mean (mm)	Std. Dev.	n	%Reduction
Control	4.22	0.185	18
1.0	4.28	0.135	7	-1.2
1.8	4.14	0.216	10	2.0
3.2	4.22	0.149	10	0.2
5.6	4.18	0.180	10	1.1
10	4.24	0.116	9	-0.4

Table7          
Effect Parameters

Parameter	Loading rate (mg/L)
NOELR for reproduction	10
NOELR for mortality	10
NOELR for growth	10

 

Table8          
Exposure parameters

 

Condition		Range
pH		7.6-8.3
Temperature measured in test vessels (°C)		19-20
Temperature measured continuously in control vessel (°C)		19-21
Dissolved oxygen concentration (mg O2/L)		7.6-9.8
Hardness (mg CaCO3)		161-179
Total Dissolved Organic Carbon content (mg/L)		0.31
Light intensity (Lux)	Start test	754-844
	End test	762-809

 

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In conclusion, under the conditions of the present study Optimol Additiv 309 did not affect reproduction, survival and growth of Daphnia magna at a loading rate of 10 mg/L after 21 days of exposure (NOELR).

Executive summary:

The objectiveof the test was to evaluate the effects of Optimol Additiv 309 on the mobility, the growth and the reproductive capacity of Daphnia magna. For this purpose, test organisms were exposed to aqueous solutions containing the test item at various concentrations. The time of the first production of young, the number of young born, immobility and other signs of intoxication observed were compared with corresponding parameters in the controls.

The study procedures described in this report were based on the OECD guideline for Testing of Chemicals: Guideline No. 211, 2012. In addition,the procedures were designed to meet the OECD guidance document number 23, 2018.

The batch of Optimol Additiv 309 tested was a pasty brown liquid UVCB substance not completely soluble in test medium at the loading rates tested.

Water Accommodated Fractions (WAFs) were individually prepared at loading rates of 1.0, 1.8, 3.2, 5.6 and 10 mg/L and used as test concentrations.

 

The reproduction test was performed in a semi-static system, which included 10 vessels per test concentration and 20 vessels for an untreated control group. Each of the vessels contained one neonate (<24h old) Daphnia magna in 50 ml test medium. The study duration was 21 days and the test solutions were renewed three times a week. The daphnids were fed on a daily basis with a Chlorella pyrenoidosa suspension. Every workday the condition of the parental daphnids was recorded, during the reproduction phase the number of living offspring, immobile young and appearance of unhatched (aborted) eggs was recorded. At the end of the test the length of the surviving parental daphnids were measured.

During the study samples for analyses were taken at the beginning and the end of one interval of 72 hours and three intervals of 48 hours.

Samples taken during this study were analysed for two components (i.e. m/z 325.3 and m/z 589.7). The calculated Time Weight Average (TWA) concentrations based on the first component increased with increasing loading rate indicating proper preparation of WAFs. The measured concentrations of m/z/ 589.7 showed high variability and no relation with the nominal loading rate. Because the test item is a UVCB substance with low solubility, the effect parameters were expressed in terms of loading rates initially prepared as recommended in OECD Guidance Document 23 (2018).

The study met the acceptability criteria prescribed by the study plan and was considered valid.

Parental mortality reached 10% in the control treatment and the highest loading rate. Three daphnids did not survive exposure in the lowest WAF. No mortality was observed in the WAFs prepared at 1.8, 3.2 and 5.6 mg/L. Hence, the mortality of the parental daphnids was not treatment related. Therefore, the effects on reproduction were calculated based on number of offspring produced by surviving parental daphnids.

On average, 142 offspring were produced per surviving daphnid in the control treatment (13 %CV). In two groups statistically significant reduction of reproduction was observed (15 and 18% in WAFs prepared at 1.8 and 5.6 mg/L, respectively). However, these effectsa coul dnot be explained by any of the measured components. For this reason, and the fact that at the highest loading rate, i.e. the regulatory limit loading rate, no significant effect was observed, it was considered justified to set the NOELR for reproduction on 10 mg/L.

The effects on growth ranged from 2.0% reduction to 1.2% increase and were not dose-related. Statistical analysis showed that none of these effects were significant.

Effect parameters (mg/L) obtained in this study are summarized in the table below

Parameter	Loading rate (mg/L)
NOELR for reproduction	10
NOELR for mortality	10
NOELR for growth	10

 

In conclusion,under the conditions of the present study Optimol Additiv 309 did not affect reproduction, survival and growth of Daphnia magna at a loading rate of 10 mg/L after 21 days of exposure (NOELR).