N,N',N'',N'''-tetrakis(4,6-bis(butyl-(N-methyl-2,2,6,6-tetramethylpiperidin-4-yl)amino)triazin-2-yl)-4,7-diazadecane-1,10-diamine

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:

From Mar. 3, 1999 to May 18, 2000

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

GLP-guideline study with restrictions - the mortality in the controls was 20% (parent animals) - the analytical recoveries of the test substance concentration of freshly prepared WAF solutions revealed strong variations - dose-response relationship inconsistent

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

During the prolonged toxicity study sampling for analysis was performed on the freshly prepared solutions on days 0, 6, 14 and 19. On days 2, 8, 16 and 21 samples were taken from the 48-hour spent solutions. Additionally samples were taken from the fresh and 72-hour old solutions of the treatment control on day 17.
The volume of the samples was 500 ml which were directly transferred to a separation funnel after centrifuging.

Vehicle:

no

Details on test solutions:

All test solutions were prepared separately and mixed with test medium by magnetic stirring for a defined period. The test product was assumed to be hydrolytically stable. Subsequently, the test solutions were allowed to stabilize. The water fractions were then centrifuged (at 30,000 g for 15 minutes) to remove any undissolved particles. The resulting water phases were defined as water accommodated fractions (WAFs) prepared at a nominal loading in mg/l.


It was decided to renew the test solutions after every 48 hours to maintain the exposure as constant as possible. As a consequence, the period of stirring prior to the renewal of the test solutions was set at 48 hours. A stabilisation period of ca. 2 to 4 hours preceded the centrifuging of all solutions to separate the water phase from the undissolved fraction of the test substance. The WAFs at 0.3 and 1.0 mg/l were prepared in total volumes of 5000 ml by mixing these volumes with 1.5 and 5.0 mg of test substance, respectively. The WAFs at 3,10, 30 and 100 mg/l were prepared in total volumes of 2000 ml by mixing these volumes with 6.0, 20, 60 and 200 mg of test substance, respectively. The actually weighed amounts did not deviate by more than 3%
from the nominal values, except in one case where 5.52 mg was weighed to prepare the WAF at 1.0 mg/l in 5000 ml.

After stabilisation the volumes necessary for centrifuging were taken from the centre part of the dispersions. When sampling for analysis was necessary, volumes of 1500 ml were taken from these water fractions and separated in volumes of 250 ml to be centrifuged (30,000 g). The supematants were pooled before taking three samples of 500 ml. One sample was used for immediate analysis. The other sample was used to fill the vessels used for exposure (ca. 50 ml
per vessel). The third sample was used for incubation under test conditions without organisms present. When no sampling for analysis was necessary, volumes of 500 ml were taken from the water fractions and separated in volumes of 250 ml to be centrifuged (30,000 g). The supernatant was used to fill the vessels used for exposure ot the parental daphnids (ca. 50 ml per vessel).

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. Protoplasms 154, 25-33).



Controls
1. Test medium without test substance or other additives (blank control).
2. Test medium without test substance or other additives but treated in the same way as the solutions treated with test substance in silanised glass ware (treatment control).

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Strain/clone: Straus, 1820
- Age of parental stock (mean and range, SD): maximum age of the cultures 4 weeks. For the test selection of young daphnids with an age of <24 hours.
- Breeding start of each batch: With newborn animals, i.e. less than 3 days old, by placing them individually in 50 ml M4-medium. The medium was renewed three times a week. Feeding: daily a suspension of Chlorella pyrenoidosa
- Feeding during test: At each renewal defined volumes of different Chlorella pyrenoidosa suspensions were added as feed for the daphnids. The daily ration was 100,000 cells/ml/day corresponding with a ration of ca. 0.2 mg C/daphnid/day.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Hardness:

250 mg/ml expressed as CaCO3

Test temperature:

19.9 and 21.3 °C

pH:

within the range of 7.0 to 8.6 throughout the study

Dissolved oxygen:

oxygen concentration in all test solutions remained > 6.0 mg/l during the exposure period

Salinity:

freshwater

Nominal and measured concentrations:

Water accommodated fractions prepared at 0.3, 1, 3, 10, 30 and 100 mg/l.

0.3 mg/L (nominal) 0.07(0.04-0.13) mg/L (measured)
1 mg/L (nominal) 0.17(0.11-0.26) mg/L (measured)
3 mg/L (nominal) 0.37(0.26-0.51) mg/L (measured)
10 mg/L (nominal) 0.85(0.59-1.22) mg/L (measured)
30 mg/L (nominal) 1.83(1.13-2.96) mg/L (measured)
100 mg/L (nominal) 4.24(2.21-8.15) mg/L (measured)

Details on test conditions:

TEST SYSTEM
- Test vessel: Volume: 50 ml (4.5 x 0.38 cm), all-glass (silanised with dichlorodimethylsilane) covered with a Perspex plate.

- Renewal rate of test solution: Every 48 hours, except on day 17, when solutions were renewed after 72 hours.

- No. of organisms per vessel: Exposed individually from the start of the 21-day exposure period.

- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 20
- No. of vessels per vehicle control (replicates): 20

Preliminary tests indicated that the actual concentration in the water phase was stable after 24 hours. Available data showed that adsorption to glass was a major cause for loss of test substance concentration. Less adsorption was observed to polypropylene. Therefore, all glassware to be used for the study was silanised applying a 2% dichlorodimethylsilane solution in hexane. The glassware was dried in a oven prior to silanisation. Subsequently, the glassware was rinsed with small amounts of the silanisation solution. Then, the glassware was rinsed with hexane to remove any surplus of dichlorodimethylsilane. The hexane was removed by evaporation applying air under pressure. Then all glassware was rinsed three times with deionized water and three times with M7 medium.

EXPERIMENTAL DESIGN:
The daphnids were exposed individually from the start of the 21-day exposure period. Ten neonate daphnids (<24h old) were used per test concentration, while the control group (0 mg/l) consisted of 20 daphnids. Each test vessel contained ca. 50 ml of test solution. Shortly after preparation of the test media the daphnids were introduced into the test medium.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: ultra-pure water (tap water purified by reverse osmosis and subsequently passed over activated carbon and ion-exchange cartridges)

- Intervals of water quality measurement: At the start of the test and just before and after each renewal in one of the vessels of all test solutions. (pH, dissolved oxygen and temperature)

OTHER TEST CONDITIONS
- Photoperiod: 16 hours light, 8 hours dark
- Light intensity: 500 to 700 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- Parental Daphnia
Immobility and mortality: Every workday and at each renewal, the number of living, immobile and dead parental daphnids were recorded. Dead daphnids were removed when observed.
Presence of eggs in the brood pouch: Every workday and at each renewal.

- Fl generation
Appearance first brood: When observed.
Newborn daphnids: Every workday and at each renewal the number of newborn young was counted and the condition of the young recorded. Thereafter the young were removed.
Presence of unhatched eggs: When observed.
Incidence of immobility: When observed.

DATA HANDLING:
Evaluation of the results and calculation of toxicity parameters was based on the nominal loadings of the test substance and on the estimated exposure concentrations, which were based on linear regression analysis of the geometric means of the concentrations measured in the fresh and spent solutions at the intervals selected for sampling. The values for reproduction observed at various concentrations of the test substance in water were expressed as the cumulative mean number of living young per parent. The values for reproduction at each concentration were expressed as percentage of those recorded in the controls on the various days of recording.
- Statistical analysis:
The results of reproduction and body length recorded on day 21 were tested for normality and for homogeneity of variance. Furthermore, these data were statistically tested using an ANOVA test followed by the Tukey's test of multiple comparisons and the Dunnet T-test or the Bonferroni's TTest (SAS V. 6.12).
The overall threshold level of effect (overall LOEC) and the overall NOEC were determined on basis of these statistics. The EC50 for reproduction was calculated using linear regression analysis.

Reference substance (positive control):

no

Duration:

21 d

Dose descriptor:

other: EC0

Effect conc.:

3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks:

parental

Remarks on result:

other: WAF

Duration:

21 d

Dose descriptor:

other: EC100

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks:

parental

Remarks on result:

other: WAF

Key result

Duration:

21 d

Dose descriptor:

other: EC0

Effect conc.:

0.37 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks:

parental

Remarks on result:

other: 95% CL 0.26 - 0.51 mg/L

Remarks:

WAF

Duration:

21 d

Dose descriptor:

other: EC100

Effect conc.:

4.24 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks:

parental

Remarks on result:

other: 95% CL 2.21 - 8.15 mg/L

Remarks:

WAF

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

12 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: 95% CL 2.1 - 72 mg/L

Remarks:

WAF

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

0.98 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: 95% CL 0.28 - 3.4 mg/L

Remarks:

WAF

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: WAF

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

0.85 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: 95% CL 0.59 - 1.22 mg/L

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: WAF

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.37 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: 95% CL 0.26 - 0.51 mg/L

Remarks:

WAF

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

> 30 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth

Remarks on result:

other: WAF

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

> 1.83 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth

Remarks on result:

other: 95% CL 1.13 - 2.96 mg/L

Remarks:

WAF

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

30 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth

Remarks on result:

other: WAF

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

1.83 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth

Remarks on result:

other: 95% CL 1.13 - 2.96 mg/L

Remarks:

WAF

Details on results:

- Mortality of parent animals: Parental mortality was 20% in both controls.
No aborted eggs were observed in any of the concentrations tested. Also, no dead offspring was recorded. Exposure to the nominal loadings of 10 and 30 mg/l induced significant inhibition of the reproductive capacity of the parental daphnids. At these levels and especially at 100 mg/l., algal cells used for the feeding of the daphnids were observed clotting. Hence, the effect on reproduction observed at these levels may also be a result of an adverse test substance effect on the quality of the food. Furthermore, test substance particles remained in the supernatant after centrifuging and caused rather high recoveries in some of the samples exceeding the expected solubility level of <= 2 mg/L. These observations would then relate to indirect effects, instead of a direct toxic effect.

In order to calculate an EC50 value for effects on reproduction, the calculated reproduction rates of the individual daphnids were compared to the average reproduction rate in the treatment control (i.e. 176 young per parent). Thus, the percentages for inhibition in reproductive rate relative to the treatment control were calculated. Calculation of the EC50 value was based on linear regression analysis of the percentages of inhibition in reproduction versus the corresponding nominal concentrations of the test item. The linear relation was calculated by applying the least squares method. The EC50 value with 95% confidence intervals was calculated by means of interpolation from the regression line. Note that the calculation includes the data on the cumulative young per parent recorded on day 12 at the nominal loading of 100 mg/l, the last day that offspring was found before all parental daphnia had died or completely immobilised.

Since the impurities might have a higher solubility level than the main component, the composition of the test product in the water phase could differ markedly from the original composition of the test product when solutions were prepared at levels above the solubility limit. The concentrations of the impurities in the test solutions were not analysed in the test solutions. Given the observed effects and the number of impurities, the WAF approach was appropriate for this Daphnia study and effect concentrations could be based the loading rates.

See attached supplementary information on test conditions, analytics, data handling, observations and results.

Reported statistics and error estimates:

EC50 for reproduction was calculated by linear regression analysis.
LOEC and NOEC for reproduction were calculated by ANOVA-Dunnet T-test and Tukey's test; P=0.05.
LOEC and NOEC for growth were calculated by ANOVA-Dunnet T-test, Bonferroni T-test and Tukey's test; P=0.05.

Measured concentrations of the test substance in water samples

 

There was not much consistency between concentrations measured at the various intervals between renewals. However, the concentrations measured in the fresh and old solutions within the periods of renewals were consistent in most cases. This indicated that repeatability of the preparation of the test concentrations was very limited, but that the concentrations measured remained rather stable during the periods between renewals. Incidentally, test substance particles remained in the supernatant after centrifuging, even when the supematants had been decanted and centrifuged a second time. This may account for the rather high recoveries found in some of the samples, with some of them exceeding the expected solubility level of <= 2 mg/l, especially in the WAFs prepared at 30 and 100 mg/l.

 

Table: The average measured concentrations of the selected intervals between the renewal of the test solutions and the estimated exposure concentrations

 

WAF at x mg/l	Geometric average concentration				Exposure in mg/l
	Day 0-2	Day 6-8	Day 14-16	Day 19-21
0.3	0.111	0.077	0.056	0.044	0.07(0.04-0.13)
1	0.231	0.322	0.062	0.085	0.17(0.11-0.26)
3	0.541	1.052	0.734	0.188	0.37(0.26-0.51)
10	1.330	0.583	1.320	0.789	0.85(0.59-1.22)
30	0.448	0.641	2.575	8.195	1.83(1.13-2.96)
100	5.416	2.733	-	-	4.24(2.21-8.15)

 

 

Parental mortality

 

Four out of the twenty parental daphnids died during the test period in both controls (see Table). Hence, parental mortality was 20% in the controls. On day 13 all parental daphnids had died at 100 mg/l. At the lower concentrations of 3 to 30 mg/l, parental mortality varied from 0 to 20%. However, 30 % had died at 0.3 mg/l, while only 50% survived at 1.0 mg/l. Hence, the mortality of the parental daphnids was not clearly treatment related.

 

There was no statistical difference between any of the groups and the treatment control according to the Tukey test, Bonferroni t-test or Dunnet t-test (P=0.05).

 

Table: Cumulative mortalities of daphnids during 21 days exposure

Nominal concentration (mg/L)	Measured concentration (mg/l)	Number of daphnids exposed	Mortality (%)
0 (blank)	-	20	20
0 (treatment)	-	20	20
0.3	0.07(0.04-0.13)	10	30
1	0.17(0.11-0.26)	10	50
3	0.37(0.26-0.51)	10	0
10	0.85(0.59-1.22)	10	20
30	1.83(1.13-2.96)	10	20
100	4.24(2.21-8.15)	10	100

 

 

The average cumulative number of young per female in the controls after 21 days was > 60 (185 + 31.9 and 176 ± 25.5). Exposure to the nominal loadings of 10 and 30 mg/l induced significant inhibition of the reproductive capacity of the parental daphnids (ANOVA, Tukey test and Dunnet ttest, P=0.05). At these levels, algal cells used for the feeding of the daphnids were observed clotting. Hence, the effect on reproduction observed at these levels could also be a result of an adverse test substance effect on the quality of the food. This would then relate to an indirect effect, instead of a direct toxic effect. No aborted eggs were observed in any of the concentrations tested. Also, no dead offspring was recorded.

 

For further information please see also #1 "2019 Excerpt OECD 211 NOTOX Project 255037.pdf" in the attachments.

 

Description of key information

NOECreproduction = 3 mg/L (WAF, loading rate)

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

NOEC

Effect concentration:

0.37 mg/L

Additional information

A chronic study investigated the effects on survival and reproductive performance in Daphnia magna during 21 days of exposure according to OECD guideline no. 211 (1998) in a semistatic study with media renewal every 48 hours. Due to the low water solubility and the impurity profile of the substance water accomodated fractions were prepared. Daphnia were exposed to water accommodated fractions at 100, 30, 10, 3, 1, 0.3 mg/l loading after separation of undissolved substance by centrifugation. Due to the high adsorption tendency test vessels were silanised. Based on the analytical results it can not be excluded that undissolved particles were present in the test solutions.

Since the impurities might have a higher solubility level than the main component, the composition of the test product in the water phase could differ markedly from the original composition of the test product when solutions were prepared at levels above the solubility limit. The concentrations of the impurities in the test solutions were not analysed in the test solutions. Given the observed effects and the number of impurities, the WAF approach was appropriate for this Daphnia study and effect concentrations could be based the loading rates.

The overall NOEC after 21 days is 3 mg/l loading.