Population and biomarker responses of Daphnia magna towards anticholinesterase exposures

Figures & data

Table 1. Sampling schedules

Purpose	Volume (mL/system)	Day after insecticide administration
For insecticide residue analysis	100–500	0, 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39
For measuring content of NAGase	20	−12, −9, −6, −3, 0, 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39 d
For animal counting and ChE activity measurements	100–500	−12, −9, −6, −3, 0, 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39

Table 2. Measured concentration of chlorpyrifos in dilutions^a

Days after dose administration	Treatment (μg/L)
	0.02	0.10	0.50	2.50	12.50
0	0.014	0.077	0.371	1.908	9.631
3	0.011	0.040	0.206	1.123	5.590
6	0.009	0.020	0.098	0.641	2.127
9	0.007	0.010	0.059	0.251	1.053
12	ND	0.007	0.027	0.090	0.594
15	ND	ND	0.016	0.061	0.423
18	ND	ND	0.011	0.038	0.262
21	ND	ND	0.008	0.013	0.107
24	ND	ND	ND	0.010	0.053
27	ND	ND	ND	0.008	0.024
30	ND	ND	ND	0.008	0.024
33	ND	ND	ND	ND	0.010
36	ND	ND	ND	ND	ND
39	ND	ND	ND	ND	ND

^aData listed in table are result of measurements from pooled samples. ND: Not detectable, i.e. lower than 0.01 μg/L.

Figure 1. Population dynamics of neonates in chlorpyrifos exposures.

Each column indicated an average of three measurements. The vertical bar indicated standard deviation of the three measurements. Each of the systems (including CK, which was free from insecticide administration) received acetone of 588 μL.

Figure 2. Population dynamics of adults in chlorpyrifos exposures.

Each column indicated an average of three measurements. The vertical bar indicated standard deviation of the three measurements. Each of the systems (including C K, which was free from insecticide administration) received acetone of 588 μL.

Figure 3. Inherent activity of ChE in adults in different exposure of chlorpyrifos.

Each column indicated an average of three measurements. The vertical bar indicated standard deviation of the three measurements. Each of the systems (including CK, which was free from insecticide administration) received acetone of 588 μL.

Figure 4. Apparent activity of ChE in adults in different exposure of chlorpyrifos.

Each column indicated an average of three measurements. The vertical bar indicated standard deviation of the three measurements. Each of the systems (including CK, which was free from insecticide administration) received acetone of 588 μL.

Figure 5. Immuno-reactive content of NAGase in media.

Each column indicated an average of three measurements. The vertical bar indicated standard deviation of the three measurements. Each of the systems (including CK, which was free from insecticide administration) received acetone of 588 μL.

Table 3. Result of multiple comparisons for each of the test indexes^a

Days after dose administration	Test indexes
	Population density	Inherent activity of ChE	Apparent activity of ChE	Immuno-reactive content of NAGase
−12	A/A/A/A/A/A/	A/A/A/A/A/A/	A/A/A/A/A/A/	A/A/A/A/A/A/
−9	A/A/A/A/A/A/	A/A/A/A/A/A/	A/A/A/A/A/A/	A/A/A/A/A/A/
−6	A/A/A/A/A/A/	B/B/A/AB/AB/AB/	A/A/A/A/A/A/	A/A/A/A/A/A/
−3	A/A/A/A/A/A/	A/A/A/A/A/A/	A/A/A/A/A/A/	A/A/A/A/A/A/
0	A/A/A/A/A/A/	A/A/A/A/A/A/	A/A/A/A/A/A/	A/A/A/A/A/A/
3	B/A/C/D/D/E/	A/B/C/D/E//	B/A/B/C/D//	B/A/B/B/B/C/
6	A/A/A/B/C/D/	A/B/B/C/D//	A/A/A/B/C//	A/A/A/A/B/C/
9	A/A/A/B/C/D/	A/A/B/C/D//	B/B/A/C/D//	A/A/B/A/C/D/
12	A/A/A/B/C/D/	A/A/B/B/C//	B/B/B/A/C//	A/A/A/A/B/C/
15	A/A/A/A/B/C/	A/A/B/C/C//	B/B/AB/A/AB//	A/A/B/B/B/C/
18	A/A/A/A/B/C/	A/A/A/B/C//	C/C/B/A/BC//	A/A/AB/B/B/C/
21	A/A/A/A/A/B/	A/A/A/B/C//	B/B/B/B/A//	A/A/A/A/B/C/
24	A/A/A/A/A/B/	A/A/AB/B/C//	B/B/B/B/A//	A/A/A/A/A/B/
27	A/A/A/A/A/B/	A/A/A/B/B//	B/B/B/AB/A//	A/A/A/A/A/B/
30	A/A/A/A/A/B/	A/A/A/A/B//	A/A/A/A/A//	A/A/A/A/A/B/
33	A/A/A/A/A/B/	A/A/A/A/B//	A/A/A/A/A//	A/A/A/A/A/B/
36	A/A/A/A/A/B/	A/A/A/A/A//	A/A/A/A/A//	A/A/A/A/A/B/
39	A/A/A/A/A/B/	A/A/A/A/A//	A/A/A/A/A//	A/A/A/A/A/B/

^aEach letter or letter combination separated by “/” indicates result of a multiple comparison performed among six treatments, i.e. CK, 0.02, 0.10, 0.50, 2.50, 12.50 μg/L. Order of the letters is in accordance with order of size of value of the test indexes. The values represented by letter (or letter combination) of the same (or partly the same) are statistically the same (i.e. p ≥ 0.01), whereas the values represented by letter (or letter combination) of completely different are statistically different (i.e. p < 0.01). The letters (or letter combinations) that are different from CK are painted grey.

Table 4. Points of departure of chlorpyrifos for each of the test indexes

		Test indexes
Endpoints		Population density	Inherent activity of ChE	Apparent activity of ChE	Immuno-reactive content of NAGase
NOEC (µg/L)	Nominal	0.10	?	0.10	0.10
	TWA^a	0.026	?	0.019	0.019
LOEC (µg/L)	Nominal	0.50	<0.02^b	0.50	0.50
	TWA^a	0.128	<0.011 ^b	0.092	0.092
MATC^c (µg/L)	Nominal	0.224	?	0.224	0.224
	TWA^a	0.058	?	0.042	0.042
Time lasting for the effect^d (d)		Up to 12	Up to 6	Up to 18	Up to 18

^aTWA is equal to the sum of the portion of each time period multiplied by the level of chemical during the time period divided by the period up to which the effect lasts. The level of chemical during the time period is counted as geometric average in that period.

^bThis value is indefinite because no test had been performed with nominal concentration lower than 0.02 µg/L.

^cMATC is the geometric average of NOEC and LOEC.

^dAccording to Van Den Brink et al. (2009), the impact could not be recognized unless it lasted for a period of two successive times or more.

Table 5. Some points of departure of chlorpyrifos reported by other researchers

No.	Condition	Population or community in concern	Nominal NOEC/LOEC (μg/L)	Time lasting for the effect	TWA-based NOEC/LOEC (μg/L)	Reference
1	Outdoor	Moina micrura	0.1/1	9 weeks		Daam et al., 2008
2	Outdoor	Daphnia group galeata	0.1/1	43 d	0.0144/0.121	López-Mancisidor et al., 2008
3	Indoor	Cladocera community^a	?/<5	>35 d		Liu et al., 2013
4	Indoor	Simocephalus sp.	?/<2.3	>56 d		Xiao et al., 2017
		Scapholeberis sp.	?/<2.3	>56 d
5	Indoor	Simocephalus vetulus	0.005/0.05	>14 d	/<0.0326	Daam & Van Den Brink, 2007
		Chydorus sphaericus	0.005/0.05	>14 d
6	Indoor	Daphnia group galeata	1/10	>35 d	0.0551/0.551	Van Wijngaarden et al., 2005
		Simocephalus vetulus	0.1/1	21 d	0.00919/0.0919
		Daphnia group galeata	0.1/1	>28 d	0.00735/0.0735

^aA community that consisted of Simocephalus sp., Moina sp., Chydorus sp., and Alona sp.

Table 6. Regressive equations established between population and ChE (3 to 21 d after dose administration)

	y	x	n	Logistic equation	R^2	P
A	Population density from 3 to 21 d (number/L)	Inherent activity from 3 to 21 d (U/µg)	105 ^a	y = 213.939372/(1 + EXP(1.199015–3.303077x))	0.5568	0.0000
B	Population density from 3 to 21 d (number/L)	Apparent activity from 3 to 21 d (U/µg)	105 ^a	y = 180.945731/(1 + EXP(1.289695–410.040519x))	0.4792	0.0000
C	Population density of 18-d average from 3 to 21 d (number/L)	Inherent activity of 18-d average from 3 to 21 d (U/µg)	5 ^b	y = 194.181822/(1 + EXP(0.946662–3.666915x))	0.9897	0.0103
D	Population density of 18-d average from 3 to 21 d (number/L)	Apparent activity of 18-d average from 3 to 21 d (U/µg)	5 ^b	y = 648.734214/(1 + EXP(1.934723–80.564603x))	0.9097	0.0903
E	Decline of 18-d average in population density from 3 to 21 d (%)	Decline of 18-d average in inherent activity from 3 to 21 d (%)	5 ^b	y = 25.046257/(1 + EXP(4.124150–0.142111x))	0.9810	0.0190
F	Decline of 18-d average in population density (%)	Decline of 18-d average in apparent activity from 3 to 21 d (%)	5 ^b	y = 23.160000/(1 + EXP(11.710990–2.593855x))	0.9701	0.0299
G	Decline of 18-d average in population density from 3 to 21 d (%)	Inherent activity of 18-d average from 3 to 21 d (U/µg)	5 ^b	y = 25.045463/(1 + EXP(−10.088550 + 15.733024x))	0.9810	0.0190
H	Decline of 18-d average in population density from 3 to 21 d (%)	Apparent activity of 18-d average from 3 to 21 d (U/µg)	5 ^b	y = 23.160000/(1 + EXP(−255.751281 + 24202.098357x))	0.9638	0.0362

^aConstituting 5 levels × 7 dates × 3 replications. ^b Each is an average of 7 dates × 3 replications.

Figure 6. Status of population and that of ChE 3 to 21 d after dose administration.

A: y – Population density from 3 to 21 d (number/L); x – Inherent activity from 3 to 21 d (U/µg). B: y – Population density from 3 to 21 d (number/L); x – Apparent activity from 3 to 21 d (U/µg). C: y – Population density of 18-d average from 3 to 21 d (number/L); x – Inherent activity of 18-d average from 3 to 21 d (U/µg). D: y – Population density of 18-d average from 3 to 21 d (number/L); x – Apparent activity of 18-d average from 3 to 21 d (U/µg). E: y – Decline of 18-d average in population density from 3 to 21 d (%); x – Decline of 18-d average in inherent activity from 3 to 21 d (%). F: y – Decline of 18-d average in population density from 3 to 21 d (%); x – Decline of 18-d average in apparent activity from 3 to 21 d (%). G: y – Decline of 18-d average in population density from 3 to 21 d (%); x – Inherent activity of 18-d average from 3 to 21 d (U/µg). H: y – Decline of 18-d average in population density from 3 to 21 d (%); x – Apparent activity of 18-d average from 3 to 21 d (U/µg).

Table 7. Regressive equations established between population and NAGase (3 to 21 d after dose administration)

	y	x	n	Logistic equation	R^2	P
A	Population density from 3 to 21 d (number/L)	NAGase content from 3 to 21 d (µg/mL)	126 ^a	y = 168.645199/(1 + EXP(6.647678–11.412781x))	0.7989	0.0000
B	Population density of 18-d average from 3 to 21 d (number/L)	NAGase content of 18-d average from 3 to 21 d (µg/mL)	6 ^b	y = 180.027737/(1 + EXP(5.306716–6.970247x))	0.9945	0.0004
C	Decline of 18-d average in population density from 3 to 21 d (%)	Decline of 18-d average in NAGase content from 3 to 21 d (%)	6 ^b	y = 100.636599/(1 + EXP(3.719743–0.101274x))	0.9940	0.0005

^aConstituting 6 levels × 7 dates × 3 replications. ^b Each is an average of 7 dates × 3 replications.

Figure 7. Status of population and that of NAGase 3–21 d after dose administration.

A: y – Population density from 3 to 21 d (number/L); x – NAGase content from 3 to 21 d (µg/mL). B: y – Population density of 18-d average from 3 to 21 d (number/L); x – NAGase content of 18-d average from 3 to 21 d (µg/mL). C: y – Decline of 18-d average in population density from 3 to 21 d (%); x – Decline of 18-d average in NAGase content from 3 to 21 d (%).

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