Glycine, N,N'-1,2-ethanediylbis-, reaction products with formaldehyde, iron chloride (FeCl3) and phenol, potassium salts

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:

2020-03-18 to 2020-04-08

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Version / remarks:

2012

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Test item: HBED-Fe

CAS-No: 1463474-95-4

Batch number: FC-C 11140

Purity (certified): 96.6% (UVCB)

Water: 3.4%

Appearance: Dark red-brown, microgranules (solid)

Water solubility: 50 – 100 g/L

Expiry date: 2022-08-31

Recommended storage: Keep in a dry place. Store at room temperature in the original container. Keep container tightly closed.

Storage conditions 18 – 25 °C, dark, in tightly closed original container

Analytical monitoring:

yes

Details on sampling:

Determination of the test item
The limit concentration and the control were analyzed via HPLC-DAD at 3 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) as well as in the old media at the end of an exposure-renewal interval (48 or 72 hours).
The samples were analyzed under GLP with an HPLC-DAD method which has been implemented under non-GLP and documented finally in the GLP raw data. The HPLC-DAD method was validated.

Sampling for the analytical monitoring
At the start of the exposure intervals on day 0, 9 and 14, samples of the fresh media were taken after preparation of the limit test item concentration and analyzed. At the end of the exposure intervals on day 2, 12 and 16 (48 or 72 hours), samples of the old media were taken directly from the test vessels. For the longest exposure-renewal interval of 72 hours, samples were taken at the start (0 hours) and at the end of the exposure interval (72 hours) once within the test period.

Vehicle:

no

Details on test solutions:

Definitive Test
Based on the results of the non-GLP preliminary range finding test, the definitive test was performed under semi-static conditions with a renewal of the test solutions every two to three days.

Preparation of the stock solution
A stock solution of 100 mg/L of the test item was prepared with the culture medium for each exposure interval. An appropriate amount of the test item was weighed out and transferred into a measuring flask with an appropriate amount of dilution water. The stock solution was shaken manually at room temperature.

Test item concentration
A limit concentration of 100 mg/L of the test item was tested.

Control
10 replicates of dilution water without test item

Test method
The study was performed under semi-static conditions with a renewal of the test solutions every two or three days (i.e. on Monday, Wednesday and Friday).

Test organisms (species):

Daphnia magna

Details on test organisms:

Test system
The test species was Daphnia magna STRAUS, obtained from continuous laboratory cultures.

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

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, 16 hours illumination; light intensity of max. 1500 lx

Culture medium
Elendt M4, according to OECD 211, Annex 2, is used. The composition of the culture medium is presented in Table .

Feeding of the culture stocks
The culture daphnids are fed at least 5 times per week ad libitum with a mix of unicellular green algae, e.g. Pseudokirchneriella subcapitata and Desmodesmus subspicatus. The algae are cultured at the test facility.

Composition of the Culture Medium Elendt M4
according to OECD 211, Annex 2
Component Concentration [mg/L]
CaCl2 x 2 H2O 294
MgSO4 x 7 H2O 123
KCl 5.80
NaHCO3 64.8
Na2SiO3 x 5 H2O 7.47
NaNO3 0.274
KH2PO4 0.143
K2HPO4 0.184
Na2EDTA x 2 H2O 2.50
FeSO4 x 7 H2O 0.996
H3BO3 2.86
MnCl2 x 4 H2O 0.361
LiCl 0.306
SrCl2 x 6 H2O 0.152
RbCl 0.0710
NaBr 0.0160
Na2MoO4 x 2 H2O 0.0615
CuCl x 2 H2O 0.0168
ZnCl2 0.0130
CoCl2 x 6 H2O 0.0100
KI 0.00325
Na2SeO3 0.00219
NH4VO3 0.000575
Thiaminhydrochloride 0.075
Cyanocobalamin 0.0010
Biotin 0.00075
pH 8.2  0.8

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

21 d

Hardness:

Total Hardness [mg/L] as CaCO3
Test group fresh old fresh old fresh old
Day 0 Day 2 Day 9 Day 12 Day 14 Day 16
100 mg/L 280 266 268 260 280 272
Control 274 267 258 249 280 270
The total hardness was above 140 mg/L as CaCO3.

Test temperature:

Temperature [°C]
Test group fresh old fresh old fresh old
Day 0 Day 2 Day 9 Day 12 Day 14 Day 16
100 mg/L 20.5 21.3 20.2 20.9 20.0 20.8
Control 20.2 21.3 20.2 21.0 18.9 20.9

pH:

pH-Values
Test group fresh
Day 0 old
Day 2 fresh
Day 9 old
Day 12 fresh
Day 14 old
Day 16
100 mg/L 7.78 7.38 7.74 7.08 7.57 7.08
Control 7.98 7.53 7.78 7.16 7.80 7.12
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:

Dissolved Oxygen Concentration [mg/L]
Test group fresh old fresh old fresh old
Day 0 Day 2 Day 9 Day 12 Day 14 Day 16
100 mg/L 8.81 5.73 8.97 4.81 8.54 5.61
Control 8.66 6.25 8.88 8.58 9.02 8.41
The dissolved oxygen concentration was above 3 mg/L.

Nominal and measured concentrations:

Nominal: 100 mg/L

Details on test conditions:

Test method
The study was performed under semi-static conditions with a renewal of the test solutions every two or three days (i.e. on Monday, Wednesday and Friday).

Test duration
21 days

Test vessels
Glass beakers (5 (ID) x 8 (H) cm), 100 mL capacity, loosely covered with watch glasses

Test volume
50 mL

Test medium
Same composition as the culture medium

Number of daphnids and 10 daphnids in 10 replicates were used for the limit concentration
replicates and the control with one daphnid per replicate, which was held individually.

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 the following period 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 was equivalent to the culture medium.

Application
50 g test solution per replicate were weighed out into each test vessel. 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 were renewed every 2 or 3 days. For this purpose, a second set of test vessels was filled with the freshly prepared test solutions and the daphnids were transferred by pipette (see ‘Application’).

Feeding
Daily; Pseudokirchneriella subcapitata (0.581 - 1.22 mL) and Desmodesmus subspicatus (0.485 - 0.666 mL) suspension was provided as food corresponding to 0.2 mg C per Daphnia and day. The density of the algae suspension varied, but it was the same for all test groups on each feeding day.

Test temperature (target)
The temperature was in the range of 18 – 22 °C and did 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.

Reference substance (positive control):

no

Key result

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Remarks:

Analytical recovery >80% nominal

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Remarks:

Analytical recovery >80% nominal

Basis for effect:

reproduction

Key result

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

> 96.6 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Remarks:

Analytical recovery >80% nominal

Basis for effect:

reproduction

Remarks on result:

other: Active ingredient content 96.6%

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Remarks:

Analytical recovery >80% nominal

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

other: LC20

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

21 d

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

21 d

Dose descriptor:

other: LC100

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Details on results:

see aný other information on materials and methods

Reported statistics and error estimates:

Statistical evaluation
The NOEC and LOEC for reproduction and adult mortality were determined with ToxRat Professional software. Significant differences of reproduction were determined in comparison to the control using statistical standard procedures as normality test (Shapiro-Wilk’s test), variance homogeneity test (Levene’s test), two-sample t-test for reproduction and Fischer’s Exact Binomial Test for adult mortality. Prior to running the two-sample t-test a normality test and a variance homogeneity test were performed. P values of the normality and the variance homogeneity test were 0.01. The a-value = 0.01 for the normality test and the variance homogeneity test.
For effects on reproduction, the juveniles per parent at the start of the test excluding accidental and inadvertent mortality (reproductive output) were counted.

Determination of the EC- and LC-values
The EC10 / 50 -values for the reproductive output and the LC20 / 50 /100 - value for adult mortality were empirically derived from the observed data.

Calculation of the intrinsic rate of natural increase (IR):
The intrinsic rate of natural increase (IR) was calculated for the intrinsic rate survived parental daphnids with the EULER-LOTKA-equation with ToxRat Professional software.
The intrinsic rate of natural 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. Clearly, the latter is not sustainable and ultimately will lead to extinction.

Software
All data are computer-processed and rounded for presentation. Consequently, minor variations may occur from the original figures if manual calculations based on the original figures are made subsequently. Calculations were made using the following software:
- Excel, MICROSOFT CORPORATION
- ToxRat Professional, Version 3.3.0, TOXRAT SOLUTIONS GMBH

Statistical Analyses

Number of Living Juveniles per Survived Parental Daphnid

(based on the nominal concentrations of the test item [mg/L])

Shapiro-Wilk´s Test on Normal Distribution

Normality check was passed (p > 0.01).

Levene´s Test on Variance Homogeneity (with Residuals)

The Levene test indicates variance homogeneity (p > 0.010). Variance homogeneity check was passed (p > 0.01).

Normal-distribution and variance-homogeneity requirements are fulfilled.

A parametric multiple test is advisable.

Two-sample t-test Procedure

Two-sample t-test Procedure with cumulative offspring per survived parent at 21 d: Two-sample comparison of treatments with "Control". Significance was Alpha = 0.050, one-sided smaller; Mean: arithmetic mean; n: sample size; s: standard deviation; MDD: minimum detectable difference to Control (in percent of Control); t: sample t; p(t): probability of sample t for ; the differences are significant in case p(t) <= Alpha  (The residual variance of an ANOVA was applied; df = N - k; N: sum of treatment replicates n(i); k: number of treatments).

      

Treatm. [mg/L]      Mean              s   df     %MDD              t           p(t)       Sign.           

            Control       108.0         7.32                                                                                     

              100.0       109.8         7.32   16         -5.6         0.52        0.693               -           

+: significant; -: non-significant

There is no statistically significant difference between Control and 100 mg/L. A NOEC of 100 mg/L is suggested by the program.

Adult Mortality

(based on the nominal concentrations of the test item [mg/L])

Fisher`s Exact Binomial Test

Fisher`s Exact Binomial Test with immobility at 21.000 d: Two-sample comparisons between sample and control (Alpha is 0.050; one-sided greater); Ho (no effect) is accepted, if the probability p(exact) > Alpha;' p(exact) is the probability that the increase in category "Immobile"  observed in the treatment(s) is due to chance.

Treatm.[mg/L] Introduced         Mobile     Immobile  % Immobility p(exact)           sign.

            Control             10                    9                  1               10.0                                     

                 100             10                    9                  1               10.0        0.763                  -

+: significant; -: non-significant

There is no statistically significant difference between Control and 100 mg/L.

Table12:   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	-	-	21	-	-	-	27	-	M						60
2	-	-	-	-	-	-	10	-	-	18	-	-	-	27	-	-	28	-	-	22	-	105
3	-	-	-	-	-	-	12	1	-	19	-	-	-	26	-	-	23	-	-	27	-	108
4	-	-	-	-	-	-	-	12	-	-	21	-	-	18	-	-	23	-	-	-	28	102
5	-	-	-	-	-	-	-	13	-	-	22	-	-	23	-	-	28	-	-	30	-	116
6	-	-	-	-	-	-	-	16	1	-	21	-	-	21	-	-	28	-	-	29	-	116
7	-	-	-	-	-	-	-	12	-	-	24	-	-	19	-	-	26	-	-	27	-	108
8	-	-	-	-	-	-	13	-	-	14	-	-	-	20	-	-	23	-	1	27	-	98
9	-	-	-	-	-	-	-	15	-	-	20	-	-	21	-	-	29	-	-	30	-	115
10	-	-	-	-	-	-	-	12	-	-	17	-	-	20	-	-	29	-	-	26	-	104
Based on all parental daphnids introduced at the start of the exposure:																						1032
Based on parental daphnids survived until the end of the test:																						972
Mortality of Parental (N)	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	1	1	1	1	1	1	1
Mortality of Parental [%]	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	10	10	10	10	10	10	10

 

-       = no juveniles observed

M     = dead parental daphnid

Table13:   Observations in the Nominal Test Item Concentration of 100 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	-	-	13	-	-	-	26	-	-	24	-	-	27	-	103
2	-	-	-	-	-	-	10	-	-	13	1	-	25	-	-	23	-	-	-	32	-	104
3	-	-	-	-	-	-	15	-	-	13	4	-	27	-	-	27	-	-	-	30	-	116
4	-	-	-	-	-	-	0	10	1	-	24	1	1	27	-	-	20	-	-	32	-	116
5	-	-	-	-	M																	0
6	-	-	-	-	-	-	-	7	-	-	14	-	-	19	-	-	23	-	-	34	-	97
7	-	-	-	-	-	-	-	17	-	-	17	1	-	22	-	-	31	-	-	32	-	120
8	-	-	-	-	-	-	15	-	-	17	-	-	19	-	-	-	27	-	-	35	-	113
9	-	-	-	-	-	-	-	9	-	-	15	1	-	17	-	-	27	-	-	34	-	103
10	-	-	-	-	-	-	-	13	-	-	16	-	-	24	-	-	26	2	-	35	-	116
Based on all parental daphnids introduced at the start of the exposure:																						988
Based on parental daphnids survived until the end of the test:																						988
Mortality of Parental (N)	0	0	0	0	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1
Mortality of Parental [%]	0	0	0	0	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10

 

-       = no juveniles observed

M     = dead parental daphnid

¹⁾      = pale parental daphnid

Table14:   pH-Values

Test group	fresh Day 0	old Day 2	fresh Day 9	old Day 12	fresh Day 14	old Day 16
100 mg/L	7.78	7.38	7.74	7.08	7.57	7.08
Control	7.98	7.53	7.78	7.16	7.80	7.12

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.

 

Table15:   Dissolved Oxygen Concentration [mg/L]

Test group	fresh	old	fresh	old	fresh	old
	Day 0	Day 2	Day 9	Day 12	Day 14	Day 16
100 mg/L	8.81	5.73	8.97	4.81	8.54	5.61
Control	8.66	6.25	8.88	8.58	9.02	8.41

The dissolved oxygen concentration was above 3 mg/L.

 

Table16:   Total Hardness [mg/L] as CaCO₃

Test group	fresh	old	fresh	old	fresh	old
	Day 0	Day 2	Day 9	Day 12	Day 14	Day 16
100 mg/L	280	266	268	260	280	272
Control	274	267	258	249	280	270

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

 

Table17:   Temperature [°C]

Test group		fresh		old		fresh		old		fresh		old
		Day 0		Day 2		Day 9		Day 12		Day 14		Day 16
100 mg/L		20.5		21.3		20.2		20.9		20.0		20.8
Control		20.2		21.3		20.2		21.0		18.9		20.9

 

Validity criteria fulfilled:

yes

Conclusions:

The effects on reproduction were evaluated based on the reproduction per survived parent animal, since this parameter is the ecologically most relevant response variable and is required by the OECD test guideline 211 (2012), when no significant trend in mortality is detected. The overall effect threshold for effects of the test item under the test conditions was 100 mg/L (NOEC) and > 100 mg/L (LOEC) based on the nominal concentrations of the test item HBED-Fe. The test material is concluded non-toxic to Daphnia magna.
 

Executive summary:

A Daphnia magna reproduction test (semi-static, 21 d) of the test item HBED-Fe (batch number: FC-C 11140) was conducted at the test facility according to OECD 211 (2012) from 2020-03-18 to 2020-04-08.

Test species was Daphnia magna STRAUS. Ten daphnids, held individually, were used per limit concentration and control. At test start the daphnids were < 24 hours old. The study was carried out under semi-static conditions with a medium renewal three times per week (i.e. on Monday, Wednesday and Friday). The aim of the test was to assess the effects on the reproduction capacity and other test item-related effects or parameters such as the intrinsic rate of the natural increase, first appearance of living juveniles, occurrence of aborted eggs and stillborn juveniles, adult mortality, body length and dry weight of the parental daphnids.

The test item HBED-Fe is a Dark red-brown, microgranules (solid). The nominal limit concentration 100 mg/L HBED-Fe was selected based on the results of a preliminary range finding test (non-GLP).

A stock solution with a nominal concentration of 100 mg/L (96.6% based on active ingredient) was prepared..

The test item concentrations of HBED-Fe were analytically verified via HPLC-DAD in the fresh media at the start of the exposure-renewal intervals on days 0, 9, 14 (0 hours) and in the old media at the end of the exposure intervals on days 2, 12, 16 (aged 48 or 72 hours, respectively) in the limit concentration with surviving daphnids) and in the control.

The measured concentrations of the test item in fresh media at the start of the respective exposure-renewal intervals (0 hours) were in the range of 98 to 100% of the nominal concentrations. At the end of the respective exposure-renewal intervals (48 and 72 hours), the measured test item concentrations in the old media were in the range of 93 to 95% of the nominal concentrations.

The measured test item concentrations remained stable within ± 20% of the nominal concentrations over the test period. This indicates that the test item concentrations were successfully maintained for the duration of the test.

Since the measured test item concentrations were within ± 20% of the nominal concentrations, the nominal test item concentrations were used for the evaluation of the effect-concentrations.

The environmental conditions were within the acceptable limits. The validity criteria of the test guideline were met. To be valid, the maximum control mortality rate at females must not exceed 20% and the mean offspring number of the survived females at day 21 must be at least 60.0. In the present test, the mortality was 10% and the mean offspring number of the survived females was 108 in the control.

In Limit tests, no trend in mortality can be determined. Thus, according to OECD 211 it is required to report the effects for the cumulative offspring per survived parent provided these values are lower than in the cumulative offspring per introduced parent.

Endpoints for Reproduction and Mortality (based on the nominal test item concentrations)

Effect values                     HBED-Fe Nominal test item concentrations [mg/L]

EC10 Reproduction                     > 100

EC50 Reproduction                     > 100

LOEC Reproduction                    > 100

NOEC Reproduction                   ≥ 100

LC20 Adult mortality after 21 days  > 100

LC50 Adult mortality after 21 days       > 100

LC100 Adult mortality after 21 days       > 100

LOEC Adult mortality after 21 days       > 100

NOEC Adult mortality after 21 days       ≥ 100

Description of key information

No effects on any endpoint were detectable up to 96.6 mg a.i./L of HBED on Daphnia magna. The test material is therefore concluded as non-toxic to aquatic invertebrates.

A similar result was observed for EDDHMA-Fe. This substance was tested upto a concentration of 1000 mg/L and resulted in a NOEC of 320 mg/L The parental mortality NOEC was 1000 mg/L

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

96.6 mg/L

Additional information

The acute toxicity to Daphnia magna was also performed with an analogue material EDDHMA-Fe according to OECD guideline 202. An EC50 of 628 mg/L based on reproduction and an EC50 of greater than 1000 mg/L based on parental mortality was determined. The animals were exposed also exposed to EDDHMA-Fe for 21 days in a chronic static test system (Bogers, 1996) where a NOEC of 320 mg/L was derived.