Quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06-04-2020 - 25-06-2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Test item
Quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides
Batch number SPA19005
CAS No. 61789-77-3; 68391-05-9
Purity (certified) 95.6% m/m
Density 880 kg/m3 at 20°C
Appearance Yellow, viscous liquid, paste
Water Solubility Dispersible
Stability under test conditions Not specified
Expiry date 2021-03-12
Recommended storage Keep in a well ventilated place.

Analytical monitoring:

yes

Details on sampling:

Determination of the test item
All concentration levels and the control were analyzed via LC-MS/MS at 3 intervals during the test (once within a period of 7 days) in the fresh media at the start of an exposure-renewal interval (0 hours) as well as in the old media at the end of an exposure-renewal interval (24 hours).
The samples were analyzed under GLP with an LC-MS/MS method which has been implemented under non-GLP and documented finally in the GLP raw data. The LC-MS/MS method were validated.
The peak distribution of the WAF was analyzed in fresh prepared medium in the highest test item concentration, once during the test. Therefore a high resolution MS (Q-ToF) was used.
Once during the test, all old media samples from one interval were centrifuged to separate the algae from the water phase. Algae and water were analyzed separately to determine the truly dissolved test item fraction and the fraction adsorbed to algae. The centrifuge tubes were rinsed with the appropriate test solutions to minimize adsorption to the walls of the centrifuge tubes.

Sampling for the analytical monitoring
At the start of the exposure-renewal intervals, samples of the fresh media were taken after preparation of all concentrations and analyzed.
At the end of the exposure-renewal intervals (24 hours),

Vehicle:

no

Details on test solutions:

Definitive Test
Based on the results of the non-GLP preliminary range finding test mentioned in Annex II, the definitive reproduction test was carried out with five concentrations of the test item. The definitive test was performed under semi-static conditions with a daily renewal of the test solutions and a coating phase of the test vessels. The stability of the test item over an exposure interval was confirmed in a non-GLP stability test.

Water Accommodated Fraction
Water accommodated fractions (WAFs) were prepared because the test item is a UVCB substance with compounds of different water solubility. This procedure is in accordance with the OECD guidance document No. 23 (2019).
Using this approach, aqueous media were prepared by mixing the test item with water for a prolonged period sufficient to ensure equilibration between the test item and the water phase.

Preparation of the water accommodated fractions
Five water accommodated fractions (WAFs) were prepared 24 ± 2 hours before the start of the exposure and each renewal day with nominal loading rates of the test item in the range of 0.0205 - 0.0512 - 0.128 - 0.32 - 0.800 mg test item/L, set up in a geometric series with a factor of 2.5.
The procedure according to ASTM D6081 (2019) as described below was carried out for each exposure-renewal interval the same way to achieve a constant quality during the course of the study.
Each water accommodated fraction (WAF) was prepared separate. The test item was homogenized after melting at 70 °C for at least 2 hours before preparation of the WAFs. For each loading rate an appropriate amount of the test item was placed on a glass slide with methanol. The methanol was evaporated. The glass slide with the test item was inserted in a glass flask with an appropriate amount of dilution water (see Table 2). A slow stirring procedure was applied for 24 ± 1 hour at 20 ± 2 °C. The magnetic stirrer bar was placed with a fish-clip® system a few centimeters above the bottom of the flask to prevent direct contact with the test item on the bottom. After a separation phase of 1 hour, the aqueous phase of the WAF was removed by siphoning (from the approximate middle of the glass flask). The first 25 mL were discarded.
The WAFs were checked via laser beam (Tyndall effect) for undissolved test item (formation of an emulsion). No presence of undissolved test item during the test was observed. The resulting water accommodated fractions (WAF) were used in the test.
For the preparation of the water accommodated fractions a stirring phase of 24 hours was found to be suitable. Longer stirring phases led to a decrease of the loading in the WAFs (< LCL). Therefore, a stirring phase of 24 hours is chosen for the definitive test.
 
Test loading
Per definition of the WAF all terms related to concentration level are given as loading rates because partly dissolved compounds and mixtures cannot be related to concentrations.
The loading rates are based on the results of a preliminary range finding test (non-GLP, open system with headspace).

Control
10 replicates of dilution water without test item

Test organisms (species):

Daphnia magna

Details on test organisms:

Test system
The test species was Daphnia magna STRAUS, obtained from continuous laboratory cultures. The daphnids of the laboratory culture originated from clone 5.

Reason for the selection of the test system
Daphnia magna is the preferred species in accordance with the test guideline and is bred at the test facility.

The Daphnia magna culture has shown to meet the validity criteria for the reproduction test of a mean of ≥ 60 living juveniles per survived parent animal when cultured under conditions described.

Origin
Institut für Wasser-, Boden- und Lufthygiene (WaBoLu),
14195 Berlin, Germany

Breeder
Noack Laboratorien GmbH,
Käthe-Paulus-Str. 1, 31157 Sarstedt, Germany

Culture
In glass vessels (2 - 3 L capacity) with approximately 1.8 L culture medium, at 20 +/- 2°C, in an incubator, 16 hours illumination; light intensity of max. 1500 lx.

Culture medium
Elendt M4, according to OECD 211, Annex 2 is used.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Hardness:

The total hardness was above 140 mg/L as CaCO3.
For details see "Any other information on results incl. tables" below.

Test temperature:

The temperature was within the range of 20 ± 2 °C.
For details see "Any other information on results incl. tables" below.

pH:

The pH-values should be in the range of 6 – 9. Significant deviations ≥ 1.5 units between the initial and final pH-values were not observed.
For details see "Any other information on results incl. tables" below.

Dissolved oxygen:

The dissolved oxygen concentration was above 3 mg/L.
For details see "Any other information on results incl. tables" below.

Nominal and measured concentrations:

nominal loading rates 0.0205 - 0.0512 - 0.128 - 0.320 - 0.800 mg/L (separation factor 2.5), corresponding to the time weighted mean measured test item concentrations 0.0130 – 0.0270 – 0.0780 – 0.148 – 0.310 mg/L.

Details on test conditions:

Test duration 21 days

Test vessels and pre-treatment
Glass beakers (5 (ID) x 8 (H) cm), 100 mL capacity, loosely covered
with watch glasses were used.
A coating phase (saturation of the test container) was carried out. The test containers were pre-treated with the appropriate test solution for at least 12 hours under test conditions. Before the start of the exposure and each renewal, the test container were emptied and refilled with freshly prepared test solutions.

Test volume
50 mL

Test medium
Same composition as the culture medium

Number of daphnids and replicates
10 daphnids and 10 replicates were used for all WAFs and the control. 1 daphnid was held individually per replicate.

Age of the daphnids
Less than 24 hours old daphnids from a healthy stock were used for at the start of the exposure the study. To achieve this, juvenile daphnids were removed from the culture vessels at the latest 24 hours before the start of the exposure and discarded. The juveniles born within this period of max. 24 hours preceding the exposure were used for the test. No first brood progeny was used for the test.

Acclimatization
Acclimatization was not necessary, because the composition of the dilution water is equivalent to the culture medium.

Application
50 g test solution per replicate were weighed into the test vessels. This corresponds to 50 mL. The daphnids were transferred in a small amount of dilution water or test solution by pipette.

Renewal of the test solutions
The test solutions and the control were renewed daily. For this
purpose, the freshly prepared test solutions were filled into a second set of test vessels and the daphnids were transferred by pipette (see ‘Application’).

Feeding
Daily feeding with unicellular green algae, e.g. Pseudokirchneriella subcapitata and Desmodesmus subspicatus, max. 0.2 mg C / daphnia and day

Test temperature (target)
The temperature should be in the range of 18 – 22 °C and should not vary by more than 2 °C.

Light intensity (target)
Max. 1500 lx

Photoperiod
16/8 hours light/dark cycle

Aeration
Test vessels were not aerated during the test.

Preliminary Range Finding Test (non-GLP)
Stability Test (non-GLP)
A WAF with a nominal loading of 2.00 mg/L was prepared. After 0, 24, 48 and 72 hours of incubation under test conditions samples were taken and analyzed via LC-MS.
For the definitive test semi-static conditions with a daily renewal of the test solutions was found to be suitable. Longer exposure intervals led to a decrease of the loading in the WAF. Therefore, a exposure interval of 24 hours is chosen for the definitive test.

Range Finding Test (non-GLP)

A non-GLP preliminary range finding test was conducted at the test facility as a shortened reproduction test (at least until observation of the first brood in all test groups) under semi-static conditions with daily renewal of the test solutions over a period of 10 days. A control and three water accommodated fractions (WAFs) with loadings levels of 0.010, 0.100 and 1.00 mg test item/L were tested in the range finding test. The preliminary range finding test was conducted under diffuse light conditions (light intensity of max. 1500 lx, 16/8 hours light/dark cycle). Five daphnids, one daphnia per replicate, were exposed to each loading level and the control.
The loading levels were analytically verified via LC-MS/MS in the fresh media (WAF, before distribution to the test vessels) at the start of the exposure and in the old media (test vessels containing daphnia and algae) at the end of an exposure-renewal interval. The analytical results are presented in Table 34.
No measurable adsorption to the glass surfaces of the test vessels was found.
For the preparation of the water accommodated fractions a stirring phase of 24 hours was found to be suitable. Longer stirring phases led to a decrease of the loading in the WAFs (< LCL). Therefore, a stirring phase of 24 hours is chosen for the definitive test.

Reference substance (positive control):

no

Key result

Duration:

21 d

Dose descriptor:

EL50

Effect conc.:

0.208 mg/L

95% CI:

ca. 0.147 - ca. 0.296

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: 0.0199 (CI: 0.141-0.283) mg a.i./L

Key result

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

0.11 mg/L

95% CI:

ca. 0.087 - ca. 0.14

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: 0.1042 (CI: 0.0831-0.1338) mg a.i./L

Key result

Duration:

21 d

Dose descriptor:

EL10

Effect conc.:

0.109 mg/L

95% CI:

ca. 0.081 - ca. 0.147

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: 0.1042 (CI: 0.0773-0.141) mg a.i./L

Key result

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

0.071 mg/L

95% CI:

ca. 0.058 - ca. 0.087

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: 0.0677 (CI: 0.055-0.0.083) mg a.i./L

Key result

Duration:

21 d

Dose descriptor:

other: NOEL

Effect conc.:

0.021 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: 0.0196 mg a.i./L

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.013 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: 0.0124 mg a.i./L

Details on results:

Reproductive Output

The number of juveniles in all replicates of the control and the test groups was counted and recorded every day.

The average number of living juveniles at the end of the test after 21 days per introduced parental female daphnid was 117 in the control group and thus the validity criterion for the reproduction of a mean of ≥ 60 living offspring per survived parent animal in the control was met.

Accidental and inadvertent mortality were not observed. Since the Cochran-Armitage trend test revealed a significant increasing trend (p ≤ 0.05) in adult mortality, the evaluation was done based on the cumulative offspring per introduced (female) parent.

A statistically significant reduction of the reproductive output in comparison to the reproductive output in the control was determined at the nominal loading rates of 0.0512 to 0.800 mg/L. Therefore, the LOEL was determined to be 0.0512 mg/L and the NOEL 0.0205 mg/L for the cumulative offspring per introduced parent. The LOEC was determined to be 0.0270 mg/L and the NOEC 0.0130 mg/L for the cumulative offspring per introduced parent.

The determination of the EL/EC10-value and its 95% confidence limits was carried out by point estimates from the 3 parametric normal CDF. The EL10 for the test item was 0.109 mg/L and the EL50 0.208 mg/L per introduced parent. The EC10 for the test item was 0.0708 mg/L and the EC50 0.110 mg/L per introduced parent.

 
Adult Mortality

Accidental and inadvertent mortality as defined in the OECD test guideline were not observed. In the control no mortality of the introduced adults was observed.
The test item induced adult mortality of 100% after 21 days in the nominal loading rates 0.320 and 0.800 mg/L, 30% adult mortality in the loading rate 0.128 mg/L. No mortality was observed at the nominal loading rates 0.0205 and 0.0512 mg/L. The Cochran-Armitage test procedure revealed a significant increasing trend (p ≤ 0.05) in adult mortality. A statistically significant dose-response was detected. The determination of the LL/LC50-value was carried out by probit analysis. The confidence limits were estimated from the observed data. The LL50 was 0.140 mg/L (confidence limits: 0.128 – 0.320 mg/L). The LOELAdult mortality was 0.128 mg/L. The NOELAdult mortality was 0.0512 mg/L. The LC50 was 0.0863 mg/L (confidence limits: 0.0780 – 0.148 mg/L). The LOECAdult mortality was 0.0780 mg/L. The NOEC Adult mortality was 0.0270 mg/L.


Intrinsic Rates of Natural Increase (IR)

The intrinsic rate of population increase is a measure of population growth which integrates reproductive output and age-specific mortality. In steady state populations it will be zero. For growing populations it will be positive and for shrinking populations it will be negative.

 
First Appearance of living Juveniles

The first appearance of living juveniles was observed between days 7 and 10 at the introduced parental daphnids of the control and the nominal loading rates of 0.0205 to 0.800 mg/L of the test item.


Analytical Results

The test item concentrations of Quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides were analytically verified via LC-MS/MS three times during the test (once within a period of 7 days) in the fresh media at the start of an exposure-renewal interval (0 hours; on test days 0, 7 and 14) as well as in the old media at the end of an exposure-renewal interval (24 hours; on test days 1, 8, 15) in all WAFs and in the control.
Five WAFs were prepared and tested at nominal loading rates 0.0205 - 0.0512 - 0.128 - 0.320 - 0.800 mg/L (separation factor 2.5). All evaluations are based on the nominal loading rates of Quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides and the time weighted mean measured test item concentrations. The test item is a UVCB substance.
The measured concentration of the test item in the fresh media (0 hours) was < LOQ to 140% of the nominal test item loading and < LOQ to 83% in the aged media after 24 hours The adsorption of the test item on the feeding algae was investigated on day 15 after centrifugation. Amounts between < LOQ and 108% of nominal loading rate were measured.
 
 
Fingerprint of the test item (non-GLP)

The peak distribution of the WAF was analyzed in fresh prepared medium in the 0.320 mg/L test item concentration. An analytical standard of the test item was prepared in methanol and diluted to 160 µg/L with acetonitrile : ultra-pure water (50 : 50) containing 1% formic acid. The WAF prepared with the highest test item loading was diluted factor 2 with acetonitrile containing 2% formic acid to avoid an inhomogeneous sample. The standard dilution and the test item dilution were analytical verified via high resolution MS (Q-ToF) and evaluated by the software. The detected signals of the analytical standard and of the test item solution were compared. In both solutions 6 masses, related to the test item could be observed. No additional signals, than analyzed during the study, related to the test item could be observed in the analytical standard or test item. No shift in intensity of the different substances could be observed.


Water Quality Parameters

The measured water quality parameters (i.e. pH-value, dissolved oxygen concentration, total water hardness and water temperature) were within the acceptable limits. The incubator temperature was in the range of 19 to 20°C.

Endpoints for Reproduction and Mortality
(based on the nominal loading rates of the test item and the time weighted mean measured test item concentrations)

 

Effect values	Quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides
	based on	based on
	the nominal loading rates	the time weighted mean measured concentrations
	[mg/L]	[mg/L]
EL/EC10 Reproduction (introduced)	0.109 (Cl: 0.0809 – 0.147)	0.0708 (Cl: 0.0577 – 0.0868)
EL/EC50 Reproduction (introduced)	0.208 (Cl: 0.147 – 0.296)	0.110 (Cl: 0.0869 – 0.140)
LOEL/LOEC Reproduction (introduced)	0.0512	0.0270
NOEL/NOEC Reproduction (introduced)	0.0205	0.0130
LL/LC20 Adult mortality after 21 days	0.0512 ˂ LL20 ˂ 0.128	0.0270 ˂ LL20 ˂ 0.0780
LL/LC50 Adult mortality after 21 days	0.140 (Cl: 0.128 – 0.320)	0.0863 (Cl: 0.0780 – 0.148)
LL/LC100 Adult mortality after 21 days	0.320	0.148
LOEL/LOEC Adult mortality after 21 days	0.128	0.0780
NOEL/NOEC Adult mortality after 21 days	0.0512	0.0270

 

Stillborn Juveniles and Aborted Eggs produced by the Survived Parental Daphnids after 21 Days

 

Nominal	Number of		Total no.
loading rate
of the test item			å
[mg/L]	stillborn	aborted
	juveniles	eggs
0.800	0	0	0
0.320	72	0	72
0.128	4	26	30
0.0512	6	0	6
0.0205	0	0	0
Control	0	0	0

 

Total Body Length and Dry Weight of the Survived Parental Daphnids

Nominal	Total length of the survived parent animals [mm]										MV	N	Dry weight
loading rate													[mg]
of the test item	Replicate no.
[mg/L]	1	2	3	4	5	6	7	8	9	10	[mm]		S	MV
0.800	-	-	-	-	-	-	-	-	-	-	-	-	-	-
0.320	-	-	-	-	-	-			-	-	-	-	-	-
0.128	4.5	4.5	5.0	-	4.5	4.0	-	5.0	-	4.5	4.57	7	2.9	0.41
0.0512	4.5	4.5	5.0	4.5	4.5	5.0	5.0	5.0	4.5	4.5	4.70	10	5.5	0.55
0.0205	5.0	5.0	4.5	4.5	5.0	5.0	5.0	5.0	5.0	4.5	4.85	10	6.8	0.68
Control	4.5	5.0	5.0	5.0	5.0	4.5	5.0	5.0	5.0	5.0	4.90	10	6.0	0.60

 

Observations in the Control

 

Day	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15	16	17	18	19	20	21
Replicate No.	Number of Juveniles																					total
1	-	-	-	-	-	-	12	-	-	19	-	-	25	-	-	30	-	-	-	24	-	110
2	-	-	-	-	-	-	-	14	-	18	-	-	27	-	-	-	30	-	-	26	-	115
3	-	-	-	-	-	-	-	12	-	-	20	-	-	30	-	-	34	-	-	20	-	116
4	-	-	-	-	-	-	-	16	-	-	23	-	-	32	-	-	36	-	-	31	-	138
5	-	-	-	-	-	-	-	13	-	-	23	-	-	31	-	-	31	-	-	29	-	127
6	-	-	-	-	-	-	-	13	-	-	9	-	-	24	-	-	30	-	-	25	-	101
7	-	-	-	-	-	-	-	16	-	2	18	-	-	30	-	-	26	-	-	16	-	108
8	-	-	-	-	-	-	-	15	-	-	18	-	-	21	-	-	34	-	-	26	-	114
9	-	-	-	-	-	-	16	1	-	18	2	-	30	-	-	-	35	-	-	21	-	123
10	-	-	-	-	-	-	-	14	-	-	16	-	-	26	-	-	30	-	-	29	-	115
Based on all parental daphnids inserted at the start of the exposure:																						1167
Based on parental daphnids survived until the end of the test:																						1167
Mortality of Parental (N)	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
Mortality of Parental [%]	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0

 

Observations in the Nominal Loading Rate of 0.0205 mg/L

 

Day	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15	16	17	18	19	20	21
Replicate No.	Number of Juveniles																					total
1	-	-	-	-	-	-	-	10	-	-	26	-	-	29	-	-	29	-	-	27	-	121
2	-	-	-	-	-	-	-	7	-	-	13	-	-	30	-	-	28	-	-	22	-	100
3	-	-	-	-	-	-	15	-	-	20	-	-	32	-	-	35	-	-	35	-	-	137
4	-	-	-	-	-	-	10	-	-	15	-	-	23	-	-	25	-	-	24	-	-	97
5	-	-	-	-	-	-	-	10	1	7	-	-	28	-	-	-	34	-	-	25	-	105
6	-	-	-	-	-	-	-	10	-	-	17	-	28	-	-	-	30	-	38	-	-	123
7	-	-	-	-	-	-	-	-	14	-	-	20	-	-	20	-	-	30	-	-	27	111
8	-	-	-	-	-	-	3	10	-	9	-	-	25	-	-	-	33	-	-	23	-	103
9	-	-	-	-	-	-	-	6	-	-	17	-	22	-	-	-	30	-	-	27	-	102
10	-	-	-	-	-	-	12	-	-	15	-	-	27	-	-	31	-	-	34	-	-	119
Based on all parental daphnids inserted at the start of the exposure:																						1118
Based on parental daphnids survived until the end of the test:																						1118
Mortality of Parental (N)	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
Mortality of Parental [%]	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0

 

Observations in the Nominal Loading Rate of 0.0512 mg/L

Day	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15	16	17	18	19	20	21
Replicate No.	Number of Juveniles																					total
1	-	-	-	-	-	-	-	10	-	8	-	-	25	-	-	24	8	-	-	21	-	96
2	-	-	-	-	-	-	13	-	-	20	-	-	30	-	-	22	-	-	24	-	-	109
3	-	-	-	-	-	-	-	-	-	8	9	-	24	-	-	-	27	-	-	14	-	82
4	-	-	-	-	-	-	-	10	-	1	14	-	28	-	-	-	26	-	-	21	-	100
5	-	-	-	-	-	-	-	-	-	14	-	27	-	-	32	-	-	35	-	-	-	108
6	-	-	-	-	-	-	-	10	-	2	8	-	29	1	-	8	-	-	30	-	-	88
7	-	-	-	-	-	-	-	5	-	-	10	-	32	-	-	-	19	-	-	16	-	82
8	-	-	-	-	-	-	11	1	-	13	-	-	23	-	-	31	-	-	26	-	-	105
9	-	-	-	-	-	-	16	-	-	17	-	-	35	-	-	33	-	-	31	-	-	132
10	-	-	-	-	-	-	1	9	-	10	-	-	28	-	-	24	-	-	-	12	-	84
Based on all parental daphnids inserted at the start of the exposure:																						986
Based on parental daphnids survived until the end of the test:																						986
Mortality of Parental (N)	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
Mortality of Parental [%]	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0

 

Observations in the Nominal Loading Rate of 0.128 mg/L

Day	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15	16	17	18	19	20	21
Replicate No.	Number of Juveniles																					total
1	-	-	-	-	-	-	-	13	-	-	15	-	30	-	-	30	2	-	20	-	-	110
2	-	-	-	-	-	-	15	-	-	16	-	-	28	-	-	38	-	-	30	-	-	127
3	-	-	-	-	-	-	13	-	-	21	-	-	37	-	-	39	-	-1	-1	-	-	110
4	-	-	-	-	-	-	9	-	-	12	-	-	35	-	-	26	-	-1	2M			84
5	-	-	-	-	-	-	-	13	-	8	-	-	40	-	-	26	-	-	11	-	-	98
6	-	-	-	-	-	-	10	-	-	7	-	-	27	-	-	28	-	-	5	-	-	77
7	-	-	-	-	-	-	-	12	-	6	-	-	20	-	-	2M						40
8	-	-	-	-	-	-	-	2	-	12	-	-	35	-	-	32	-	-	-	-	-	81
9	-	-	-	-	-	-	12	-	-	14	1	-	17M									44
10	-	-	-	-	-	-	-	11	-	23	1	-	35	-	-	45	-	-	31	-	-	146
Based on all parental daphnids inserted at the start of the exposure:																						917
Based on parental daphnids survived until the end of the test:																						749
Mortality of Parental (N)	0	0	0	0	0	0	0	0	0	0	0	0	1	1	1	2	2	2	3	3	3	3
Mortality of Parental [%]	0	0	0	0	0	0	0	0	0	0	0	0	10	10	10	20	20	20	30	30	30	30

 

Observations in the Nominal Loading Rate of 0.320 mg/L

 

Day	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15	16	17	18	19	20	21
Replicate No.	Number of Juveniles																					total
1	-	-	-	-	-	-	-	7	-	12	-1	-1	231	-1	-1	-1	-1	M				42
2	-	-	-	-	M																	0
3	-	-	-	-	-1	-1	-1	8M														8
4	-	-	-	-	-	-1	-1	41	-1	M												4
5	-	-	-	-	-	-	2	-1	-1	M												2
6	-	-	-	-	-1	-	-	41	-1	-	101	-1	-1	M								14
7	-	-	-	-	-	-	1	1	-	-	-1	-1	211	-1	-1	-1	-1	M				23
8	-	-	-	-	-	-	9	-	-	1	-1	-1	171	-1	-1	-1	-1	M				27
9	-	-	-	-	-	-	-	-	-	10	-1	-1	321	-1	M							42
10	-	-	-1	-1	-1	-1	-1	41	-	131	-1	-1	261	-1	-1	-1	-1	-1	51	M		48
Based on all parental daphnids inserted at the start of the exposure:																						210
Based on parental daphnids survived until the end of the test:																						210
Mortality of Parental (N)	0	0	0	0	1	1	1	2	2	4	4	4	4	5	6	6	6	9	9	10	10	10
Mortality of Parental [%]	0	0	0	0	10	10	10	20	20	40	40	40	40	50	60	60	60	90	90	100	100	100

 

Observations in the Nominal Loading Rate of 0.800 mg/L

Day	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15	16	17	18	19	20	21
Replicate No.	Number of Juveniles																					total
1	M																					0
2	M																					0
3	M																					0
4	M																					0
5	M																					0
6	M																					0
7	M																					0
8	M																					0
9	M																					0
10	M																					0
Based on all parental daphnids inserted at the start of the exposure:																						0
Based on parental daphnids survived until the end of the test:																						0
Mortality of Parental (N)	10																					10
Mortality of Parental [%]	100																					100

 

Comparison of water quality parameters in fresh and old medium once within 7 days.

pH-Values

Test group	fresh	old	fresh	old	fresh	old
	Day 0	Day 1	Day 7	Day 8	Day 14	Day 15
Highest loading rate	7.54	6.63	7.48	7.33	7.43	8.14
Control	7.59	6.96	7.43	7.32	7.46	7.91

The pH-values should be in the range of 6 – 9. Significant deviations ≥ 1.5 units between the initial and final pH-values were not observed.

 

Dissolved Oxygen Concentration [mg/L]

Test group	fresh	old	fresh	old	fresh	old
	Day 0	Day 1	Day 7	Day 8	Day 14	Day 15
Highest loading rate	8.13	8.87	8.12	8.18	8.61	9.69
Control	8.70	6.53	8.02	7.69	8.78	6.84

The dissolved oxygen concentration was above 3 mg/L.

 

Total Hardness [mg/L] as CaCO3

Test group	fresh	old	fresh	old	fresh	old
	Day 0	Day 1	Day 7	Day 8	Day 14	Day 15
Highest loading rate	275	268	250	239	248	269
Control	276	264	248	239	252	257

The total hardness was above 140 mg/L as CaCO3.

 

Temperature [°C]

Test group	fresh	old	fresh	old	fresh	old
	Day 0	Day 1	Day 7	Day 8	Day 14	Day 15
Highest loading rate	21.0	20.3	20.6	21.2	21.6	21.5
Control	20.9	20.2	20.6	21.1	21.5	21.4

The temperature was within the range of 20 ± 2 °C.

 

 

 

 

 

Validity criteria fulfilled:

yes

Remarks:

No parent animal in the control group died. Actual mortality of the parent animals in the control group was 0%. The mean number of living offspring produced per survived parent animal at the end of the test was 117 in the control group

Conclusions:

The effects on reproduction were evaluated based on the reproduction per female parent animal inserted at the start of the exposure. The Cochran-Armitage trend-test revealed a significant trend in mortality. Thus, according to OECD 211 it is required to report the effect values for the cumulative offspring per introduced parent provided these values are lower than in the cumulative offspring per survivor. Under the study conditions,EL10 and EL50 for effects of the test substance on reproduction were determined to be0.109 mg/L(0.1042 mg a.i./L) and0.208 mg/L(0.0199 mg a.i./L) based on the nominal loading rates of the test substance and0.0708 mg/L(0.0677 mg a.i./L)and0.110mg/L(0.1042 mg a.i./L)based on the time-weighted mean measured test item concentrationsrespectively. The correspondingNOEL and LOEL were determined to be 0.0205 mg/L (0.0196 mg a.i./L) and 0.0512 mg/L (0.0489 mg a.i./L) based on the nominal loading rates of the test substance and 0.0130 mg/L (0.0124 mg a.i./L) and 0.027 mg/L (0.026 mg a.i./L) based on the time-weighted mean measured test item concentrationsrespectively

Executive summary:

A study was conducted to determine the long-term toxicity of the test substance C12-18 DAQ (95.6% active) to Daphina magna, according to the OECD guideline 211, in compliance with GLP. The test species was Daphnia magna STRAUS. Ten daphnids, held individually, were used per WAF loading rate and control. At the start of the exposure, the daphnids were less than 24 hours old. The study was carried out under semi-static conditions with daily renewal of the test solutions. The aim of the test was to assess the effects on the reproduction capacity and other test substance-related effects or parameters such as the intrinsic rate of the natural increase, the number of broods with living juveniles, the first appearance of living juveniles, the occurrence of aborted eggs and stillborn juveniles, adult mortality, body length and dry weight of the parental daphnids. The test substance is a yellow, viscous liquid, paste. The test substance is a UVCB substance with compounds of different water solubility. The aqueous media were prepared following OECD guidance document No. 23 (2019), which is specifically developed for aquatic toxicity testing of UVCBs, by mixing the test substance with water for a prolonged period sufficient to ensure equilibration between the test substance and the water phase. At the completion of mixing and following a settlement period, the WAF was separated by siphon. This procedure was followed for each renewal of the test solutions. Five WAFs were prepared and tested at nominal loading rates 0.0205 - 0.0512 - 0.128 - 0.320 - 0.800 mg/L (separation factor 2.5), corresponding to the time-weighted mean measured test substance concentrations 0.0130 – 0.0270 – 0.0780 – 0.148 – 0.310 mg/L. The WAFs were checked for any undissolved or emulsified material by the Tyndall effect, which was negative. The test substance concentrations were analytically verified via LC-MS/MS three times during the test (once within a period of 7 days) in the fresh media at the start of an exposure-renewal interval (0 hours; on test days 0, 7 and 14) as well as in the old media at the end of an exposure-renewal interval (24 hours; on test days 1, 8, 15) in all WAFs and in the control. The environmental conditions were within the acceptable limits. The validity criteria of the test guideline were met.

 

The effects on reproduction were evaluated based on the reproduction per female parent animal introduced at the start of the exposure. The Cochran-Armitage trend-test revealed a significant trend in mortality. Thus, according to OECD 211, it is required to report the effect values for the cumulative offspring per introduced parent provided these values are lower than in the cumulative offspring per survivor. All reasonable efforts were taken to produce a solution of all soluble components of the test substance in test media. Since the test substance is a multi-component mixture (UVCB), the test solution is considered water accommodated fraction (WAF). The term “loading rate” is advocated to express exposure to a WAF and is considered analogous to the nominal concentration. According to OECD guidance document No. 23 (2019), for tests with chemicals that cannot be quantified by analytical methods at the concentrations causing effects, the effect concentration can be expressed based on the nominal concentrations or loading rate (for mixtures). All evaluations are based on the nominal loading rates of the test substance and the time-weighted mean measured test substance concentrations. A summary of all endpoints based on the nominal loading rates of the test substance and the time-weighted mean measured test substance concentrations is given in Table 1.

It should be noted that the test substance is a UVCB substance with constituents of different water solubility which means that per the definition of the WAF approach, the dose-response can only be based on loading rates because partly dissolved compounds and mixtures cannot be related to concentrations. Despite this definition of WAF, the results are also presented as TWA.

 

Table 1:Endpoints for reproduction and mortality(based on the nominal loading rates of the test substance and the time-weighted mean measured test substance concentrations)

 

Effectvalues	Basedon thenominalloadingrates[mg/L]	Basedon thetime-weightedmeanmeasuredconcentrations[mg/L]
EL/EC10Reproduction(introduced)	0.109(Cl:0.0809–0.147)	0.0708(Cl:0.0577–0.0868)
EL/EC50Reproduction(introduced)	0.208(Cl:0.147–0.296)	0.110(Cl:0.0869–0.140)
LOEL/LOECReproduction(introduced)	0.0512	0.0270
NOEL/NOECReproduction(introduced)	0.0205	0.0130
EL/LC20Adultmortalityafter21days	0.0512˂LL20˂0.128	0.0270˂LL20˂0.0780
EL/LC50Adultmortalityafter21days	0.140(Cl:0.128–0.320)	0.0863(Cl:0.0780–0.148)
EL/LC100Adultmortalityafter21days	0.320	0.148
LOEL/LOECAdultmortalityafter21days	0.128	0.0780
NOEL/NOECAdultmortalityafter21days	0.0512	0.0270

 

Under the study conditions,EL10 and EL50 for effects of the test substance on reproduction were determined to be0.109 mg/L(0.1042 mg a.i./L) and0.208 mg/L(0.0199 mg a.i./L) based on the nominal loading rates of the test substance and0.0708 mg/L(0.0677 mg a.i./L)and0.110mg/L(0.1042 mg a.i./L)based on the time-weighted mean measured test substance concentrations respectively. The correspondingNOEL and LOEL were determined to be 0.0205 mg/L (0.0196 mg a.i./L) and 0.0512 mg/L (0.0489 mg a.i./L) based on the nominal loading rates of the test substance and 0.0130 mg/L (0.0124 mg a.i./L) and 0.027 mg/L (0.026 mg a.i./L) based on the time-weighted mean measured test substance concentrations respectively (Scheerbaum, 2021).

Reference 2

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From March 24, 2010 to April 15. 2010

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Supplemented natural water was used for testing. The Bulk approach was applied.

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Deviations:

yes

Remarks:

Supplemented natural water was used for testing. The Bulk approach was applied.

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Samples of all the test concentrations and the stock were taken on Day 0, 2, 5, 14, 16, and 19. Some analysis was also conducted on Day 21.
- Analysis was undertaken immediately on samples if possible to quantify the concentration of the test substance in the test solutions. If this was not possible the samples were stored together with stability samples in the refrigerator until analysis. Aqueous samples were diluted 50:50 (v/v) with
leaching solution and filtered over a 0.45 µm Pall GHP membrane acrodisc syringe filter to remove the suspended solids and or algae before analysis.

Vehicle:

not specified

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: To prepare the stock solutions for every water renewal, on average 0.0051 g of test substance was weighed, and then was added directly to approximately 80 mL of the test media. This solution was sonicated for 2 min. A homogeneous solution resulted. This solution was allowed to cool whilst stirring. The resulting solution was then transferred to a 100 mL volumetric flask and the original vessel was rinsed with test media and the volumetric flask was made up to 100 mL also with test media. A slightly opaque but homogeneous stock was achieved.
- A fresh stock solution was prepared for each solution change. Test solutions were prepared by further dilution of the stock solution with the test medium to achieve the desired test concentrations. A geometric series of concentrations were used. The ratio between two consecutive concentrations was 3.2. Test vessels were filled directly from the intermediate stocks after a short stirring period. The solutions were renewed at least three times a wk during the test. One control containing only test medium was included in the test.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Source: Dr .U. Noack-Laboratorien Käthe-Paulus- Str. 1, D-31157 Sarstedt, Germany
- Age of parental stock (mean and range, SD): Less than 24 hours old and were obtained from parent animals reproducing parthenogenically. The animals used in the test were and aged between 2-4 weeks (having previously produced at least one brood before use). Offspring were
reference tested twice a year with potassium dichromate

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Post exposure observation period:

All adults were inspected under magnification for abnormalities. Individuals were measured and dry weight was determined.

Hardness:

Ca – 40.0 mg/L
Mg – 1.43 mg/L
ºdH- 5.94ºdH (

Test temperature:

20.95 -21.90 °C.

pH:

7.1- 8.9

Dissolved oxygen:

The measured oxygen concentration did not fall below: 8.4mg O2/L.

Salinity:

Not reported

Nominal and measured concentrations:

- Nominal concentrations: 0.004, 0.015, 0.048, 0.15, and 0.5 mg/L.
- Average recoveries of the test substance were >80%. Lower concentrations proved more difficult to recover and variation of results was higher.

Details on test conditions:

TEST SYSTEM
- Test vessel: Beaker
- Type (delete if not applicable): open / closed
- Material, size, headspace, fill volume: 50 mL beaker
- Aeration:
- Type of flow-through (e.g. peristaltic or proportional diluter):
- Renewal rate of test solution (frequency/flow rate):
- No. of organisms per vessel:
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10
- Biomass loading rate:
- Food: Fed a diet of 0.1 - 0.2 mg of carbon per daphnid/d in the form of the algal strain Chlorella vulgari

OTHERS:
- The strain is cultured in the ECRA Environmental Chemistry laboratory and total organic carbon content has previously been measured and logged in the GLP system. Parent animals were checked for immobilisation on at least 6 occasions per week of the test, by gently shaking the test vessel. From the day of the first brood, observations of broods (aborted, living and dead progeny) were also made in each container at each concentration. The day of brood release and the number of living and dead neonates per brood or abortions were noted. Any other abnormal observations were also recorded.
- Temperature and dissolved oxygen, were measured in the control and in the highest concentration both old and new solutions where parent
daphnids were still alive on Day 0, 5, 14, and 21. Continuous measurement of temperature in water next to the test apparatus was also conducted. The pH was measured more regularly on Day 0, 2, 5, 7, 9, 14 and 21.

Test medium
The test medium was prepared by mixing natural surface water with standard Elendt M4 media vitamins. The appropriate M4 stock was added to the natural surface water to give a concentration of 0.075 mg/L vitamin B1, 0.001 mg/L vitamin B12 and 0.00075 mg/L of Biotin in the final test media. This media was shown to provide sufficient nutrients to support good growth and reproduction of the test animals. As previously described natural surface water was used in this test in keeping with the bulk approach. Natural surface water was sampled from Heveadorp in the district of Renkum coordinates: 51º 58’N 5º48’E. The actual sample point is illustrated by an arrow and the text “hier monster genomen”. Natural surface water was frozen until use to preserve organic contents and reduce microbial activity. The dilution water was aerated before being used in the test. The air was water-saturated and purified by activated charcoal and a cotton filter.

Reference substance (positive control):

yes

Remarks:

Daphnia Culture referenced routinely with potassium dichromate.

Key result

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

ca. 1.15 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: Adult Length

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

ca. 0.5 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

reproduction

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

ca. 0.15 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: Length & Weight

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

ca. 0.5 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mortality

Results with reference substance (positive control):

- Reference tested as part of GLP maintenance twice per year with potassium Dichromate.
- Preliminary tests were conducted to establish definitive test concentrations and suitability of media to meet validity criteria was determined prior to testing.

Reported statistics and error estimates:

- The data was tested for normality using the Kolmogorov D test.
- Homogeneity of variance was confirmed by the Bartlett’s test.
- Analysis of variance using the Dunnetts test.
- The Fisher's exact test was also used for adult mortality endpoint. The calculations were conducted using Toxcalc 5.0.23 statistical software. The same statistical analysis was applied to Reproduction, length, weight and adult mortality endpoints where possible.

Results

Preliminary tests

No existing data was present on which to base the definitive test concentrations. For this reason an acute range finding was conducted in natural surface water following the relevant OECD Daphnia acute guideline and using the same stock preparation method as detailed. Five individuals were used per beaker in duplicate. The range finding results suggest that acute effect levels appear between 0.1 and 1 mg/L. As some mortality is desired in the highest concentration of the definitive chronic test 0.5 mg/L was chosen as the highest concentration. The largest factor permitted by the guidelines was applied to test a range as broad as possible to increase the chance of reaching a NOEC concentration in the definitive test.

Water quality

The observed temperature range which was continuously recorded during the test was: 20.95 -21.90°C. The measured oxygen concentration did not fall below: 8.4mg O2/L. The monitored water hardness in the test water batch was: 106 mg/L as CaCO3. (ºdH was measured and converted using a factor of 17.9 to convert to mg CaCO3/L) Light intensity was measured at the start at the end of the test on the laboratory table and was found to be 9.9 & 10.9 µmol/m2/s respectively. Nominal concentrations tested were 0.004, 0.015, 0.048, 0.15 and 0.5 mg/L. The highest 2 concentrations and the stock solution were analytically confirmed at 80 % of the nominals or greater. The remaining concentrations were detectable at fluctuating levels due to test substance binding to suspended matter.

Analytical results

The test concentrations 0.15, and 0.5 mg/L and the stock solutions were all measured at levels greater than 80% of the nominal concentrations. The 0.048 mg/L replicate fluctuated at around the 80 % recovery mark but fell slightly below this on a few occasions. The lower concentrations proved to be less stable and measured in some cases above the 80% level and in some cases considerably lower. It can be seen that at the lower concentrations the reproducibility of the analytical measurements is lower and considerably more variable than the higher concentrations. This is to be expected when measuring at such low levels with substances that adhere strongly to suspended matter. Based on the stock values, and when considering the overall analytical picture as well as the nominal amount of stock added to the replicates it can be concluded that with the exception of a few outliers (that will be discussed later) that the correct amount of test substance was added to the test vessels. Recovery levels are also high in general > 80%. The use of nominal concentrations for endpoint calculations is therefore justified. The results highlighted in red were considered to be outliers. The unexpectedly high recoveries in the 0.015 and 0.004 mg/L are caused by the lower repeatability and accuracy of the analytical method at lower concentrations. Only a slight fluctuation in the measured concentrations at 4 µg/L strongly influences the recoveries in comparison to the nominal values. Binding to suspended solids can also also result in an all or nothing analytical measurements at such low concentrations. Considering the stock analysis and the volumes added to these replicates from the stock the nominal concentrations of these results are theoretically correct but are at the limit of the analytical capability and carry therefore a greater error factor. The higher concentrations offer more stable, reproducible analytical measurements. A false measurement was also recorded in the stock measurement on day 2. This result can only be explained by the fact that the stock was sampled to early and not given enough time to stir. Such a high recovery is not possible when considering the amount of material that was added. It can be seen from the test concentrations that after homogenisation this had no negative effects on the test concentrations.

Other analytical results

Extraction of the test chemical from glass was validated in an algae test for the same test substance so was not revalidated as a part of this study. Measurements of the amount of test chemical adhering to the glass at 0.048 mg/L were conducted. 3.1% of the nominal concentration was recovered. This is negligible and glass binding will not have significantly affected the exposure. The remaining percentage of the same concentration in the water phase was 16.3 mg/L after 48 hours. This substance is not considered significantly biodegradable in the time between solution refreshments. The remainder of the test substance can be assumed to be bound to suspended solids or algae. Exposure was therefore satisfactory in keeping with the bulk approach.

Parent animal mortality

One adult daphnid died during the test period in the control. This is an acceptable mortality according to the appropriate OECD guideline. No adult daphnids died at 0.004 mg/L, 1 adult daphnid died at 0.015 mg/L, 1 adult died at 0.048 mg/L, 0 daphnids died at 0.15 mg/L and 2 daphnids died at 0.5 mg/L. A normal dose response effect was not observed for adult mortality. No significant effect in comparison to the control was detected using the Fishers exact test. The NOEC was therefore 0.5 mg/L and the LOEC >0.5 mg/L. The addition of the algae to the test replicates mitigated the effects of the test substance significantly with respect to the range finding test.

Coefficient of variation of control fecundity

The number of juvenile daphnids per replicate in each concentration was recorded. The recommended guideline criteria for the coefficient of variation (less than 25% in the control based on the number of living neonates for each parent animal surviving at the end of the test) was achieved. The mean number of neonates per surviving parent per replicate was also displayed. The total and the individual number of neonates per replicate were depicted.

Statistical results

The data on reproduction was tested for normality using the Kolmogorov D test and was found to be normally distributed. Homogeneity of variance was confirmed by the Bartlett’s test. Analysis of variance using the Dunetts test was performed on the number of living neonates per parent alive at the end of the test. Calculations were conducted using Toxcalc 5.0.23 statistical software. A NOEC for reproduction was calculated as 0.5 mg/L. A LOEC for reproduction was calculated as > 0.5 mg/L. An Ec10 for reproduction could not be calculated as a normal dose response effect was not observed in the data. Data based on adult length was also statistically analysed using the same approach as detailed above for reproduction. The calculations are presented in figure 4. A NOEC of 0.15 mg/L and a LOEC of 0.5 mg/L were calculated for the length endpoint. An Ec10 value for length was estimated without confidence limits as 1.15 mg/L using a probit plot. Weight data was also analysed in the same manner, normality of the data could not be confirmed most likely due to limited replicates but equal variances were confirmed for the data. The Dunetts test confirmed the data already observed for the length endpoint. A NOEC of 0.15 mg/L and a LOEC of 0.5 mg/L were calculated. An Ec10 for weight could not be calculated. A Fishers exact test was conducted on the adult mortality data using the same statistical software. A NOEC of 0.5 mg/L and LOEC of >0.5 mg/L were calculated.

Any other biological effects observed

Reduced feeding was clearly visible in the 0.5 mg/L replicate. This supports the statistical data from the length and weight endpoints demonstrating this concentration as a clear LOEC. No other biological abnormalities or effects were observed.

Conclusion

Based on adult mortality and reproduction no significant effects were observed. For length and weight endpoints statistically significant results were observed in the 0.5 mg/L test concentration making length and weight the most sensitive endpoints. The NOEC for adult mortality and reproduction was therefore 0.5 mg/L and the LOEC was >0.5 mg/L. The NOEC based on length and weight was 0.15 mg/L and the LOEC was 0.5 mg/L. The only Ec10 that could be calculated was for the length endpoint at 1.15 mg/L. Analysis has shown recovery in the stock and in the highest 2 concentrations and stock to be at least 80% of the nominal values. The lower concentrations could not be as accurately detected as in the higher concentrations. Recoveries as a percentage of the nominal concentrations fluctuated and fell below 80 % of the nominal values. It can be concluded however that the correct amount of test substance was added to the test vessels. This is reinforced by the amount of test chemical weighed into the stock, the analysis of the stock and the highest 2 concentrations and by the fact that the correct volumes of test substance were transferred from this stock to make the test concentrations. Due to the test substance binding strongly to suspended solids and the low detection limit required accuracy of analysis at low concentrations was limited and some outlying results were measured. Characteristics inherent to the test substance are acceptable as part of the bulk approach and would occur in the environment to a greater extent. Additional analysis demonstrated that the substance did not bind significantly to glass. The quality criteria set for the analytical method were met during measurement of the reported analytical results and the analytical method was concluded to be accurate at the time of analysis. In conclusion the test organisms were exposed to the test chemical in keeping with the bulk approach as far as was possible and the use of nominal concentrations for endpoint calculations was justified.

Amendments to and deviations from the study plan

(a) Amendments to the study plan

The study plan was amended due to a minor malfunction of the analytical apparatus. Due to a signal increase during long analytical runs calibration curves were analyzed every 6 samples to correct for this and ensure accurate analysis. The measurement of the apparatus was not affected.

(b) Deviations from the study plan

Water characterization measurements ammonium, total phosphate nitrate and total hardness were conducted as part of a GLP test but do not claim GLP compliance as the Dr Lange test system was not independently validated. Nominal concentrations were used for the calculation of effect levels in keeping with the bulk approach despite some recoveries falling below 80% of the nominal concentrations.

Quality criteria

The following quality criteria were met:

- 20% mortality occurred in the control group over the test period.

- The average number of juveniles per parent in the control was 142 after 21 days.

Natural surface water parameters

Parameter	Comment
Location	Heveadorp
Sample Date	17-3-10
Conditions when sampled	Windy ± 15ºc
Colour	clear- light yellow
Conductivity	313 µs/cm
NPOC	2.438 mg/L
TSS	18.2mg/L
Ammonium	0.041mg/L approx
Nitrate	5.79 mg/L
Total phosphate	0.083 mg/L
Total hardness	Ca – 40.0 mg/L Mg – 1.43 mg/L ºdH- 5.94ºdH (<LOD)
pH(with added Nutrients)	8.0

Analytical recovery

Concentration	T=0 (%)	T=2d (%)	T=5d (%)	T=14d (%)
Control	--	--	--	--
0.004 mg/L	78.4	103.9	64.6	210.0
0.015 mg/L	80.4	95.1	73.0	110.7
0.048 mg/L	84.7	92.2	76.3	72.1
0.15 mg/L	81.9	86.1	79.5	92.9
0.5 mg/L	80.9	85.9	79.2	86.0
Test Stock	91.8	3267.9	83.1	107.4
Concentration	T=16d (%)	T=19d (%)	T=21d (%)	Glass Extraction (%)
Control	--	--
0.004 mg/L	215.0	0.0
0.015 mg/L	82.7	46.0
0.048 mg/L	94.8	74.8	16.3	3.1
0.15 mg/L	85.8	87.7
0.5 mg/L	86.9	92.2
Test Stock	93.7	100.4

Some anomolous were results observed these are explained and justified in the report

Number of juvenile daphnids per parent alive at the end of the test at each test concentration, mean number of neonates per concentration and coefficient of variance

	Concentration (mg/L)
Rep no.	0	0.004	0.015	0.048	0.15	0.5
1	184	137	135	133	144	121
2	117	130	137	134	159	119
3	147	136	147	149	162	133
4	D	138	153	142	159	157
5	141	159	D	157	155	151
6	151	127	130	132	166	D
7	143	165	144	119	160	135
8	148	149	120	D	156	D
9	107	128	143	126	165	112
z10	140	149	129	163	167	153
Mean	142	141.8	1.375.556	1.394.444	159.3	135.125
St. dev.	216.852.484	1.315.548	1.027.267	1.451.819	6.733.828	1.710.837
CV (%)	152.713.017	927.749	7.468.016	1.041.145	4.227.136	1.266.115
Total	1278	1418	1238	1255	1593	1081

Length of adult daphnids in units

Daphnids No.	control	0.004mg/L	0.015 mg/L	0.048 mg/L	0.15mg/L	0.5mg/L
1	5.2	5.2	4.8	4.9	5.3	4.6
2	4.9	4.8	4.9	4.9	4.9	4.6
3	4.8	5.2	5.1	4.8	5.3	44
4	-	5.2	5.0	5.0	5.1	4.9
5	5.1	5.0	-	4.8	5.2	4.8
6	5.0	5.0	5.4	5.1	5.2	-
7	5.1	5.0	5.0	5.1	5.0	4.8
8	5.1	5.1	5.0	-	5.1	-
9	5.1	5.1	5.0	5.1	5.1	4.7
10	5.4	5.0	4.9	5.0	4.9	4.9

* 1 unit = 1mm

Weight of adult daphnids

Conc.( mg/L)	replicate	Weight before	No. of Daphnids’s	Total weight (g)	Weight per Daphnids (mg)
control	I	2.1782	4	2.1831	1.23
	II	2.1889	5	2.1967	1.56
0.004mg/L	I	2.1953	5	2.2011	1.16
	II	2.1929	5	2.1986	1.14
0.015 mg/L	I	2.1980	4	2.2024	1.10
	II	2.1930	5	2.1989	1.18
0.048. mg/L	I	2.1793	5	2.1853	1.2
	II	2.1544	4	2.1589	1.13
0.15mg/L	I	2.1498	5	2.1552	1.08
	II	2.1485	5	2.1542	1.14
0.5mg/L	I	2.1497	4	2.1536	0.98
	II	2.1438	4	2.1481	1.08

Validity criteria fulfilled:

yes

Conclusions:

Under the study conditions, the NOEC for reproduction, mortality, adult length and weight of the test substance for Daphnia magna were determined to be 0.5, 0.5 and 0.15 mg/L (nominal), respectively.

Executive summary:

A study was conducted to determine the long-term toxicity to Daphina magna of the test substance, C12-18 DAQ (99.6% active), according to the OECD guideline 211, in compliance with GLP.The test was

carried out according to the bulk approach using enriched natural surface water with a low Dissolved Organic Carbon (DOC) and Total Suspended Solids (TSS) content, allowing a more environmentally realistic determination of the effects of the test chemical to be made.The test organisms were exposed to test substance at nominal concentrations of 0, 0.004, 0.015, 0.048, 0.15, and 0.5 mg/L for a period of 21d. During the test organisms were exposed to the test chemical in keeping with the bulk approach as far as was possible and the use of nominal concentrations for endpoint calculations was justified. Based on the observed results the NOEC for the test substance based on reproduction, mortality, adult length and weight were determined to be 0.5, 0.5 and 0.15 mg/L (nominal), respectively. The LOEC based on reproduction and adult mortality was therefore greater than >0.5 mg/L, whereas LOEC based adult length and weight was 0.5 mg/L. The EC10 based on length was calculated as 1.15 mg/L and confidence limits could not be calculated. Mortality in the control did not exceed 20% during the test period. The average number of juveniles per surviving parent in the control was 142. Under the study conditions, the 21 d NOEC for reproduction, mortality, adult length and weight of the test substance for Daphnia magna were determined to be 0.5, 0.5 and 0.15 mg/L (nominal), respectively (Kean M, 2010).

Description of key information

Two long-term daphnia studies are available for Quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides (C12-18 DAQ). Both studies are performed semi-statically according to OECD TG 211 with specific chemical analysis (LC-MS/MS) under GLP conditions.

 

C12-18 DAQ is a multicomponent mixture (UVCB) of cationic surface-active constituents with different water solubilities. The fate of cationic surfactants in general deviates from standard chemicals. These substances are therefore considered as difficult substances for which the results of standard guideline studies are very difficult to interpret when considering them in a standard way. The reasons are the intrinsic properties like the relatively low water solubility and strong sorption to equipment and organisms. Classical ecotoxicity testing with these substances using reconstituted water often leads to test results which are poorly reproducible and are associated with high uncertainty. Because of the complex sorption mechanisms (van der Waals and Ionic mechanisms) the actual dissolved exposure concentration cannot reliably be estimated.

The two available long-term tests where therefore performed following two different approaches.

One test which is focused on determining the intrinsic toxicity of C12-18 DAQ (for C&L purposes) is performed according to the Water Accommodated Fraction (WAF) approach as described in “OECD guidance document on aqueous-phase aquatic toxicity testing of difficult test chemicals” (No. 23 Feb. 2019) with a daily refreshment of the test solutions. The term “loading rate” is advocated to express exposure to a WAF and is considered analogous to the nominal concentration.

 

The other test which is more suited to derive a realistic risk ratio for the aquatic compartment is performed according to the PECaquatic bulk/PNECaquatic bulk approach as described in ECETOC Technical Report “Environmental Risk Assessment of difficult substances” (TR 88, 2003) with a three times a week refreshment of the test solutions. The so called “Bulk approach” is used in the environmental risk assessment to cope with the earlier mentioned lack of realistic PEC estimation. Instead of using the dissolved PECwater, the Bulk concentration (dissolved + sorbed) in water is used. This bulk approach requires a PNECwater, bulk that means that testing has to use river water which contains dissolved organic carbon and suspended organic and inorganic matter, instead of reconstituted water.

Tests according to the bulk approach were thus performed because the partitioning of cationic surfactants to soil, sediment or suspended matter is rather complex which explains why there is no alternative Equilibrium Partitioning Method (EPM, di Toro, 2008) formula for these substances available yet. The use of the Bulk approach however elegantly bypasses this deficiency as it eliminates the EPM on the exposure and effect side. (More details on the Bulk approach are attached to the dossier).

 

Main difference between the two approaches lies in the preparation of the test solutions and how the results should be interpreted.

The test solutions for the WAF approach were prepared by gentle mixing the test item with test medium for a prolonged period sufficient to ensure equilibration between the test item and the water phase. At the completion of mixing and following a settlement period, the WAF was separated by siphoning. This procedure was followed for each renewal of the test solutions. Five WAFs were prepared and tested at nominal loading rates 0.0205 - 0.0512 - 0.128 - 0.320 - 0.800 mg/L (separation factor 2.5), corresponding to the time weighted mean measured test item concentrations were 0.0130 – 0.0270 – 0.0780 – 0.148 – 0.310 mg/L. No undissolved or emulsified material was observed in the WAF solutions based on the Tyndall effect check.

The results in the report are presented based on nominal Test Loadings and on Time Weighted Average (TWA) measured concentrations. The EL10/EC10 for reproduction after 21 days is 104.2/67.7 µg a.i./L. The EL50/EC50 for adult mortality after 21 days is 133.8/82.5 µg a.i./L. The TWA results are given despite the fact that per definition of the WAF, all terms related to concentration level should be given as loading rates because partly dissolved compounds and mixtures cannot be related to concentrations.

In Table 1. A summary of loading and observed concentrations is given. This table is also attached as illustration to the RSS. The nominal loading based on nominal test item concentration, active ingredient (95.6%) and based on 6 main molecular weights (ranging from C₂₄H₅₂N to C₃₄H₇₂N which represent 77.4% of the total active ingredient) are presented next to the mean measured initial and time weighted average concentrations. Because it is known that quats sorb strongly to algae (van Wijk et al., 2009) also the sorption to algae, glassware and in water was quantified. No measurable adsorption to the glass surfaces of the test vessels was found and between 78 to 93% of the measured initial concentration was found to be sorbed to algae. Only 4 – 19% was remaining in the water phase.

The daphnids in the long-term test are thus mainly exposed to C12-18 DAQ via ingestion of algae and the fact that per definition of the WAF, all terms related to concentration level should be given as loading rates because partly dissolved compounds and mixtures cannot be related to concentrations and considering the stability of the test substance and the daily renewal of the test solutions, it is considered justified to use the results based on nominal loading rates for Classification and Labeling purposes.

The test solutions for the Bulk approach were prepared by diluting a stable emulsion of 50 mg/L in test medium. In agreement with the bulk approach the test medium used was natural surface water which was characterized in detail and which was for this test enriched with standard Elendt M4 media vitamins. The following concentrations were prepared by diluting the stock solution in test medium: 0.004, 0.015, 0.048, 0.15, and 0.5 mg/L. Due to the use of non-standard test medium (natural river water) the results of bulk approach test are considered inadequate by regulators involved in C&L because they do not fulfill to the narrow criteria set to quantify the intrinsic toxicity. There is however a clear difference in the evaluation of a standard aquatic ecotoxicity test and an ecotoxicity test performed using the Bulk approach. In order to class a standard laboratory toxicity study valid, it is of particular importance that – besides information on test substance, test method/conditions and test organism used - suitable precautions are taken to prevent the loss of test substance by adsorption and that exposure concentrations are based upon measured levels of the dissolved concentration.

For ecotoxicity tests performed using the bulk approach, adsorption to suspended matter and DOC is acceptable and only adsorption to glassware should be accounted for. For a valid bulk approach test the dose-response relationship should thus be based on the sum of adsorbed and dissolved substance. Results from bulk-approach tests are therefore easier to interpret because nominal concentrations corrected for sorption to glassware can be used to quantify the dose. Because of the use of natural river water a bulk approach test is more environmentally realistic than the standard method and due to that considered to be a higher tier study. During the test, similar as in the environment, the DOC and suspended matter present will serve as passive dosing reservoirs maintaining constant exposure.

 

Droge & Goss (2013) have shown that sorption of cationic surfactants to soil and sediment is mainly driven by electrostatic interaction and to a lesser extent by hydrophobic interaction. This means that both the suspended matter and dissolved organic carbon in surface water are the key surface water properties determining the bioavailability of the test item.

The natural surface water was therefore characterized in detail and selected to contain a realistic worst-case suspended matter concentration of 15±3.5 mg/L and ± 3.5 mg/L DOC(≈NPOC). It should be noted that this composition is in perfect alignment with the risk assessment method developed by ECHA, as the concentration of suspended matter in surface water is considered to be 15 mg/L in CHESAR III for risk assessment (see ECHA’s guidance R.16, v3.0, Feb 2016, p. 88).

The resulting NOEC and EC10 values are provided in nominal concentrations corrected for 3.1% sorption to glassware because the recoveries in the fresh media were in the range of ≥80 % of the nominal values. The difference between the observed recoveries and nominal concentrations is explained by the rapid sorption of C12-18 DAQ to the river water constituents. Based on the study result, the NOEC for reproduction, mortality, adult length and weight of the test substance for Daphnia magna were determined to be 0.48, 0.48 and 0.145 mg a.i./L corrected for sorption to glassware of 3.1%, respectively.

The mitigation factor of the river water constituents in the bulk approach test can be calculated by taking the ratio between the bulk approach effect concentrations and the WAF approach effect concentrations based on loading:

NOEC bulk/ErL10 WAF = 145/104.2 = 1.4

 

 

 

• Droge, S.T.J. and Goss, K.W. 2013. Development and Evaluation of a New Sorption Model for Organic Cations in Soil: Contributions from Organic Matter and Clay Minerals. Environmental Science and Technology, 47:14233-14241.


• Di Toro, D 2008 Bioavailability of chemicals in Sediments and soils: toxicological and chemical interactions. SERDP/ESTCP Bioavailability workshop


• van Wijk, D., Gyimesi-van den Bos, M., Garttener-Arends, I., Geurts, M., Kamstra, J., Thomas, P., 2009. Bioavailability and detoxification of cationics, I. Algal toxicity of trimethylammonium salts in the presence of suspended matter and humic acid. Chemosphere 75 (3), 303–309.

 

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

EC10

Remarks:

WAF approach: The EC10 (repro) for C&L = 104.2 µg a.i./L based on loading rate and 95.6% active ingredient. Bulk approach: The NOEC (length = most sensitive endpoint) for Risk Assessment = 145 µg a.i./L (corrected for 3.1% sorption to glassware)

Effect concentration:

104.2 µg/L